Methods for treating an animal for low milk production

ABSTRACT

The present invention provides methods for treating an animal for low milk production. The methods include administering compositions including siderophore receptor polypeptides and porins from gram negative microbes, and preferably, lipopolysaccarhide at a concentration of no greater than about 10.0 endotoxin units per milliliter.

CONTINUING APPLICATION DATA

This application is a divisional of U.S. application Ser. No.10/038,504, filed Jan. 3, 2002, now abandoned, which claims the benefitof U.S. Provisional Application Ser. No. 60/259,504, filed Jan. 3, 2001,and U.S. Provisional Application Ser. No. 60/262,896, filed Jan. 19,2001, all of which are incorporated by reference herein.

BACKGROUND

The economic impact of infectious diseases in food animal production iswell appreciated. Infectious diseases reduce profits, increaseproduction costs, and endanger the overall wholesomeness of the foodproducts, as well as effect the performance, health and welfare of theanimal. This disease status can reduce the yield and quality of milkresulting in great economic loss to the dairymen. In some cases,infectious microbial diseases can cause morbidity and mortality ofnewborn, young (e.g., replacement stock) or adult animals.

The agricultural industry presently relies on antibiotic therapy andvaccines to decrease losses caused by clinical and subclinicalinfectious diseases, including gastrointestinal disease, respiratorydisease, and systemic disease. However, for some conditions, antibioticsare ineffective, may prolong the condition, or induce a carrier state.Vaccines have often proven to be an effective means of controllinginfectious diseases, but, concerns relating to adverse effects or lackof protection against multiple microbes have been a major drawback tocurrent vaccines. For instance, vaccines are available that contain oneor more immunogens against an individual genus, species, or strain ofmicrobe; however, few, if any, provide cross-protection or stimulatebroad-based immunity against multiple strains, species or genera ofmicrobe.

Vaccines containing molecules obtained from gram negative microbestypically include contaminating levels of lipopolysaccharide (LPS), acomponent of the outer membrane of most gram negative microbes. Thepresence of LPS in an injectable product can result in an inflammatoryresponse at the site of injection that can result in swelling,tenderness and often the formation of a granuloma at the site ofinjection. In rare cases, it can result in anaphylactic shock and death.This non-specific inflammatory response in a production animal canresult in significant economic losses due to increasing the likelihoodof disease by increasing the level of stress of the animal, andnegatively effecting performance characteristics of the animal. Inaddition, the formation of a granuloma at the injection site can resultin significant economic losses due to blemishes and scarring of thecarcass which are often trimmed during processing resulting in the lossof product and down grading of the carcass. While methods for removal ofLPS from compositions exist, this is often not feasible for use withvaccines that include whole cells. Moreover, due to the high costs ofremoving LPS from solutions, it is typically not economically practicalto remove LPS from vaccines for use in non-human animals.

SUMMARY OF THE INVENTION

The presence of LPS in animal vaccines has a significant economicimpact. However, the refusal of farmers to pay high fees for vaccineshas prevented the use of available, but costly, methods for LPS removal.Accordingly, there is a long standing but unresolved need for methodsfor economically producing compositions containing molecules from gramnegative microbes that contain low amounts of contaminating LPS. Thepresent invention represents an advance in the art of economicallyisolating polypeptides from gram negative microbes with low levels ofcontaminating LPS. Accordingly, the present invention provides methodsfor isolating outer membrane polypeptides. The method includes providinga gram negative microbe, disrupting the gram negative microbe in abuffer, solubilizing the disrupted gram negative microbe, and isolatingmolecules of the gram negative microbe, wherein the isolated moleculesinclude outer membrane polypeptides including at least two siderophorereceptor polypeptides (SRPs) and at least two porins, and LPS at aconcentration of no greater than about 10.0 endotoxin units permilliliter (EU/ml). During disrupting, the gram negative microbe may bepresent in the buffer at a concentration of between about 720 grams ofmicrobe per 1,000 milliliters of buffer and about 1,080 grams of microbeper 1,000 milliliters of buffer. Solubilization of the gram negativemicrobe may occur for greater than about 24 hours. Solubilization of thegram negative microbe may occur in a solution including sarcosine, wherethe ratio of the sarcosine to gram weight of disrupted gram negativemicrobe is between about 0.8 gram sarcosine per about 4.5 grams ofdisrupted gram negative microbe and about 1.2 grams sarcosine per about4.5 grams of disrupted gram negative microbe.

The present invention is also directed to a composition including atleast two SRPs isolated from a gram negative microbe, at least twoporins isolated from the gram negative microbe, and LPS at aconcentration of no greater than about 10.0 EU/ml. The composition mayfurther include a pharmaceutically acceptable carrier. The gram negativemicrobe may be an enteropathogen, preferably, a member of the familyEnterobacteriaceae, more preferably, a member of the tribe Escherichieaeor Salmonelleae, most preferably, Salmonella spp. or Escherichia coli.The at least two SRPs may have molecular weights of between about 60 kDaand about 100 kDa, and the at least two porins may have molecularweights of between about 30 kDa and about 43 kDa.

The present invention also represents an advance in the art ofstimulating immunity to multiple strains, species, or genera of microbe.Accordingly, the present invention also provides a method for inducingthe production of antibody in an animal. The method includesadministering to an animal an effective amount of a composition of thepresent invention further including a pharmaceutically acceptablecarrier, where the composition induces in the animal antibody thatspecifically binds at least one SRPs or at least one porin. The gramnegative microbe may be an enteropathogen, preferably, a member of thefamily Enterobacteriaceae, more preferably, a member of the tribeEscherichieae or Salmonelleae, most preferably, Salmonella spp. orEscherichia coli. The animal may be an avian, a bovine, a caprine, aporcine, or an ovine. When the animal is a bovine, the bovine mayexhibit a phenotype of, for instance, decreased somatic cell count,increased milk production, decreased fecal shedding, or increasedweight.

The present invention is further directed to a method for inducing theproduction of antibody in an animal, where the method includesadministering to an animal an effective amount of a composition thatincludes at least four SRPs isolated from a gram positive microbe and apharmaceutically acceptable carrier, where the composition induces inthe animal antibody to the SRP. The gram positive microbe may be amember of the family Micrococcaceae, for instance, Staphylococcusaureus. The SRPs may have molecular weights of between about 60 kDa andabout 100 kDa.

Also provided by the present invention are methods for treatingconditions in an animal, including, for instance, a high somatic cellcount, fecal shedding of a microbe in an animal's intestinal tract, lowmilk production, mastitis in a milk producing animal, and metritis in ananimal. The methods include administering to an animal having or at riskof having the condition an effective amount of a composition of thepresent invention, where the composition further includes apharmaceutically acceptable carrier.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Comparison of Salmonella isolation and serological response tovaccination in lactating cows. Percent positive isolation, percent ofvaccinated lactating cows shedding Salmonella bredeney; antibodyresponse (O.D.), optical density at 405 nm of antibody response asmeasured by ELISA. The bars correspond to the y-axis on the left(Percent Positive Isolation) and the open diamonds correspond to they-axis on the right (Antibody Response (O.D.)).

FIG. 2. The Dairy Herd Improvement Association (DHIA) somatic cell counton individual cows before and after the first vaccination. Average cellcount ×1,000, average somatic cell count times 1,000; Cows sampled,identification of each of the 51 cows; Pre-Vac, average cell count×1,000 of each cow before vaccination; Post-Vac, average cell count×1,000 of each cow before vaccination.

FIG. 3. Cumulative pounds of milk produced before and after the thirdvaccination. Bulk Tank Average (lbs), pounds of milk produced by allcows in lactation. The shaded areas “Before vaccination” and “1.2% Aftervaccination” represent the difference in percent in milk productionbefore and after the third vaccination.

FIG. 4. The cumulative rolling herd average showing pounds of milkproduced before and after vaccination in lactating cows. Pounds of milk,pounds of milk produced by all cows in lactation and averaged over theperiod of a month. Shaded area represents the rise in milk productionduring vaccination.

FIG. 5. The average monthly cost in antibiotic usage before and aftervaccination. 51% reduction refers to the reduction of the costs ofantibiotics after vaccination.

FIG. 6. The average weekly milk production between vaccinated andnon-vaccinated cows in first lactation. Weekly Milk Production/Group,weekly production of milk (in pounds) for the control group and thevaccinated cows.

FIG. 7. The average weekly milk production between vaccinated andnon-vaccinated fresh cows. Weekly Milk Production/Group, weeklyproduction of milk (in pounds) for the control group and the vaccinatedcows.

FIG. 8. The monthly average somatic cell count (DHIA) between vaccinatedand non-vaccinated cows in first lactation.

FIG. 9. The monthly average somatic cell count (DHIA) between vaccinatedand non-vaccinated fresh cows.

FIG. 10. The serological response of vaccinated steers compared tonon-vaccinated controls. Mean optical density (O.D.), 405 nm;P3-Vaccinated and P5-Vaccinated, vaccinated steers in pens 3 and 5,respectively; P4-Control and P6-Control, non-vaccinated steers in pens 4and 6, respectively; Sampling time, weeks after first vaccination.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS OF THE INVENTION

Compositions

One aspect of the present invention provides compositions includingsiderophore receptor polypeptides (SRPs) and porins obtained from amicrobe. Unless otherwise specified, the term “microbe” includes bothgram negative microbes and gram positive microbes. As used herein,“polypeptide” refers to a polymer of amino acids linked by peptide bondsand does not refer to a specific length of a polymer of amino acids.Thus, for example, the terms peptide, oligopeptide, protein, and enzymeare included within the definition of polypeptide. This term alsoincludes post-expression modifications of the polypeptide, for example,glycosylations, acetylations, phosphorylations, and the like. Apolypeptide can be produced using recombinant techniques, or chemicallyor enzymatically synthesized. Preferably, the polypeptides of thecompositions of the present invention are isolated. An “isolated”polypeptide means a polypeptide that has been either removed from itsnatural environment, produced using recombinant techniques, orchemically or enzymatically synthesized. Unless otherwise specified,“a,” “an,” “the,” and “at least one” are used interchangeably and meanone or more than one.

Gram negative microbes suitable for use in obtaining SRPs are thosecapable of producing SRPs when incubated under low iron conditions. Lowiron conditions are described herein. Such gram negative microbesinclude enteropathogens, preferably, members of the familyEnterobacteriaceae, more preferably, members of the familyEnterobacteriaceae that are members of the tribe Escherichieae orSalmonelleae, even more preferably, E. coli or Salmonella spp. Examplesof preferred enteropathogens include members of the familyEnterobacteriaceae, members of the family Vibrionaceae (including, forinstance, Vibrio cholerae), and Campylobacter spp. (including, forinstance, C. jejuni). Examples of preferred members of the familyEnterobacteriaceae include, for instance, E. coli, Shigella spp.,Salmonella spp., Proteus spp., Klebsiella spp. (for instance, Klebsiellapneumoniae), Serratia spp., and Yersinia spp. Preferred examples ofSalmonella spp. include Salmonella enterica serovars., Bredeney, Dublin,Agona, Blockley, Enteriditis, Typhimurium, Hadar, Heidelberg,Montevideo, Muenster, Newport senftenberg, Salmonella cholerasuis, andS. typhi. Salmonella enterica serovars Bredeney, Dublin and Typhimuriumare referred to herein as Salmonella bredeney, S. dublin, and S.typhimurium, respectively. Preferred examples of strains of E. coliinclude, for example, E. coli serotypes O1a, O2a, O78, and O157,different O:H serotypes including 0104, 0111, 026, 0113, 091, andhemolytic strains of enterotoxigenic E. coli such as K88⁺, F4⁺, F18ab⁺,and F18ac⁺. As used herein, the term “strain” refers to members of aspecies of microbe where the members have different genotypes and/orphenotypes. Other gram negative microbes include members of the familyPasteurellaceae, preferably Pasturella spp., more preferably, Pasturellamultocida and Pasteurella haemolytica, and members of the familyPseudomonadaceae, preferably Pseudomonas spp., most preferably,Pseudomonas aeruginosa, Yet other gram negative microbes includeActinobacillus spp., Haemophilus spp., Myxcobacteria spp.,Sporocytophaga spp., Chondrococcus spp., Cytophaga spp., Flexibacterspp., Flavobacterium spp., Aeromonas spp., among other gram-negativebacteria.

Gram positive microbes from which polypeptides may be obtained includemembers of the family Micrococcaceae, preferably, Staphylococcus spp.,more preferably, Staphylococcus aureus. Other gram positive microbesinclude members of the family Deinococcaceae, preferably, Streptococcusagalactiae, Streptococcus uberis, Streptococcus bovis, Streptococcusequi, Streptococcus zooepidemicus, or Streptococcus dysgalatiae. Othergram positive microbes from which polypeptides can be isolated includeBacillus spp., Clostridium spp., Corynebacterium spp., Erysipelothrixspp., Listeria spp., and Mycobacterium spp., Erysipelothrix spp., andClostridium spp.

These microbes are commercially available from a depository such asAmerican Type Culture Collection (ATCC). In addition, such microbes arereadily obtainable by isolation techniques known and used in the art.The microbes may be derived from an infected animal as a field isolate,and screened for production of SRPs, and introduced directly into lowiron conditions, or stored for future use, for example, in a frozenrepository at about −20° C. to about −95° C., preferably about −40° C.to about −50° C., in bacteriological media containing 20% glycerol, andother like media.

The present invention provides compositions including at least two,preferably, at least three, siderophore receptor polypeptides (SRPs).SRPs of gram negative microbes are polypeptides present in the outermembrane of gram negative microbes, and SRPs of gram positive microbesare polypeptides present in the membrane of gram positive microbes. Insome aspects of the invention, SRPs are expressed by a microbe at highlevels when the microbe is exposed to low iron conditions, and expressedat a substantially lower level when the microbe is exposed to high ironconditions. Preferably, SRPs are expressed by a microbe when the microbeis exposed to low iron conditions, and not expressed at detectablelevels when the microbe is exposed to high iron conditions. Low ironconditions and high iron conditions are described in greater detailherein. Without intending to be limited by theory, it is believed thatthe SRPs of the present compositions are receptors of iron-bindingsiderophores. Examples of siderophore receptors expressed by gramnegative microbes include, for instance, receptors for the uptake ofaerobactin, enterobactin, ferric citrate, ferrichrome, rhodotorulic, andcoprogen, as well as receptors for the transferrins (for instance theserotransferrins, lactotransferrin, and ovotransferrin), and otherbinding proteins, (see, for instance, Emery et al., U.S. Pat. No.5,830,479, and Crichton, Microbial Iron Uptake and IntracellularRelease. In: Inorganic Biochemistry of Iron Metabolism, Burgess, (ed).,Ellis Horwood Limited, Chichester, England, 59–76 (1991)).

Preferably, SRPs of the compositions of the present invention haveimmunogenic activity. “Immunogenic activity” refers to the ability of apolypeptide to elicit an immunological response in an animal. Animmunological response to a polypeptide is the development in an animalof a cellular and/or antibody-mediated immune response to thepolypeptide. Usually, an immunological response includes but is notlimited to one or more of the following effects: the production ofantibodies, B cells, helper T cells, suppressor T cells, and/orcytotoxic T cells, directed to an epitope or epitopes of thepolypeptide. “Epitope” refers to the site on an antigen to whichspecific B cells and/or T cells respond so that antibody is produced.

It is known to the art that receptors of siderophores typically includeepitopes that are conserved in the SRPs of different species anddifferent genera of microbes (see, for instance, Emery et al. (U.S. Pat.No. 5,830,479) and Example 8). For instance, antibodies produced againstan aerobactin receptor protein of one species, strain or genus of thefamily Enterobacteriaceae (for instance, E. coli, Salmonella spp., andKlebsiella spp.) have been found to cross-react with other microbeswithin the family. Species of Pseudomonas of the family Pseudomonadaceaealso express siderophore receptor proteins that can be isolated asdescribed herein and produce antibodies that cross-react with thereceptor proteins of E. coli, Salmonella spp., and Klebsiella spp.,among other members of the family Enterobacteriaceae. Moreover,antibodies produced against SRPs of Salmonella and against SRPs of E.coli have been found to cross react with the gram positive microbeStaphylococcus aureus (see Example 11).

A composition of the present invention may contain at least two,preferably, at least three, SRPs isolated from one or more genera or oneor more species of microbe. In some aspects of the present invention,preferably the SRPs of a composition are derived from multiple speciesof the same genus of microbe, or from multiple strains of the samespecies of microbe. The present invention also includes compositionsincluding SRPs isolated from at least one gram negative microbe and atleast one gram positive microbe. Preferably, the molecular weights ofSRPs, as determined by separation of the SRPs using an about 12% sodiumdodecyl-polyacrylamide gel electrophoresis (SDS-PAGE) gel under reducingand denaturing conditions, are between about 60 kDa (kiloDaltons) andabout 100 kDa, more preferably, between about 65 kDa and about 95 kDa.

Typically, different species of Salmonella each produce three SRPs.Without intending to be limited by theory, it is believed that the threeSRPs produced by Salmonella spp. are receptors for the siderophoresenterochelin, aerobactin, and ferrichrome. Preferably, SRPs obtainedfrom S. dublin and S. typhimurium are combined. Preferably, themolecular weights of SRPs isolated from Salmonella, as determined byseparation of the SRPs using an about 12% SDS-PAGE gel under reducingand denaturing conditions, are between about 60 kDa and about 100 kDa,more preferably, between about 65 kDa and about 95 kDa. More preferably,the molecular weights of SRPs isolated from Salmonella are as follows:between about 87 kDa and about 91 kDa, preferably about 89 kDa; betweenabout 82 kDa and about 86 kDa, preferably about 84 kDa; and betweenabout 69 kDa and about 75 kDa, preferably about 72 kDa.

E. coli have been found to produce 2, 3, 4, or 6 SRPs, depending on theserotype. Preferably, a composition that includes SRPs from E. coliincludes, in increasing preference, at least two, at least three, atleast four, or at least six SRPs isolated from E. coli. SRPs isolatedfrom different E. coli strains can be combined. Preferably, themolecular weights of SRPs isolated from an E. coli, as determined byseparation of the SRPs using an about 12% SDS-PAGE gel under reducingand denaturing conditions, are between about 60 kDa and about 100 kDa,more preferably, between about 65 kDa and about 95 kDa. More preferably,in a composition including SRPs isolated from an E. coli, the SRPs havemolecular weights selected from between about 91 kDa and about 93 kDa,preferably about 92 kDa; between about 88 kDa and about 90 kDa,preferably about 89 kDa; between about 82 kDa and about 86 kDa,preferably about 84 kDa; between about 76 kDa and about 80 kDa,preferably about 78 kDa; between about 73 kDa and about 75 kDa,preferably about 74 kDa; and between about 71 kDa and about 73 kDa,preferably about 72 kDa. A preferred composition that includes SRPsisolated from E. coli is isolated from the E. coli deposited with theAmerican Type Culture Collection, 10801 University Blvd., Manassas, Va.,20110-2209, USA, on Dec. 29, 1994, and designated ATCC #55652. Thedeposit was made under the Budapest Treaty on the InternationalRecognition of the Deposit of Microorganisms for the Purposes of PatentProcedure.

Field isolates of the gram positive microbe Staphylococcus aureus hasbeen found to produce at least about 4 SRPs. Preferably, when thecomposition includes SRPs from S. aureus, the SRPs are isolated from atleast one species of S. aureus, more preferably, from one species of S.aureus. Preferably, the S. aureus is isolated from an avian animalsuffering from a disease caused by S. aureus. Preferably, the molecularweights of four of the SRPs isolated from an S. aureus, as determined byseparation of the SRPs using an about 10% SDS-PAGE gel under reducingand denaturing conditions, are between about 60 kDa and about 100 kDa,more preferably, between about 65 kDa and about 95 kDa. More preferably,the molecular weights of SRPs isolated from S. aureus are as follows:between about 88 kDa and about 92 kDa, preferably about 90 kDa; betweenabout 82 kDa and about 86 kDa, preferably about 84 kDa; between about 70kDa and about 74 kDa, preferably about 72 kDa; and between about 64 kDaand about 68 kDa, preferably about 66 kDa. Preferably, the molecularweights of the other three SRPs isolated from S. aureus are betweenabout 35 kDa and about 37 kDa, preferably about 36 kDa; between about 30kDa and about 34 kDa, preferably about 32 kDa; and between about 20 kDaand about 24 kDa, preferably about 22 kDa. Preferably, an S. aureus fromwhich the SRPs are isolated is obtained from a bird, for instance achicken or a turkey, displaying symptoms of a disease caused by the S.aureus, for instance, septicemia.

Preferably, SRPs of the present compositions can be identified usingantibodies that specifically bind SRPs. As used herein, an antibody thatcan “specifically bind” a polypeptide is an antibody that interacts withthe epitope of the antigen that induced the synthesis of the antibody,or interacts with a structurally related epitope. Such antibodies can bemade using the E. coli strain having the designation ATCC #55652.Typically, ATCC #55652 is grown under low iron conditions, and SRPs areisolated from the strain as described in Example 1, or as described by,for example, Emery et al. (U.S. Pat. No. 5,830,479). Antibody is thenmade that specifically binds the SRPs using laboratory methods forproducing polyclonal and monoclonal antibodies. Such laboratory methodsare routine and known in the art (see, for instance, Harlow E. et al.Antibodies: A laboratory manual Cold Spring Harbor Laboratory Press,Cold Spring Harbor (1988) and Ausubel, R. M., ed. Current Protocols inMolecular Biology (1994)). Methods for determining whether SRPs of thepresent compositions are specifically bound by antibodies made usingSRPs isolated from ATCC #55652 are routine and known to the art, andinclude, for instance, western immunoblot and enzyme linkedimmunosorbant assay.

The compositions of the present invention also include at least twoporin polypeptides. Porin polypeptides are transmembraneous poreforming-proteins of the outer membrane of gram negative microbes. Gramnegative bacteria have a cell wall with a thin peptidoglycan membranelayer in which small hydrophilic compounds can diffuse through the outermembrane by the porin pathway. Gram positive microbes have a thickpeptidoglycan layer, which is porous and does not form a permeabilitybarrier on the surface. It has been widely accepted that gram positivebacteria do not possess well-defined pore-forming proteins as comparedto gram-negative bacteria. Nevertheless, recent evidence has shownidentification of channel-forming activity in some members of the familyCorynebacteriaceae. Unlike SRPs, the expression of porins does notchange in response to the level of iron present in the medium in which amicrobe is grown, and porin expression is typically constitutive.Without intending to be limited by theory, it is believed that theporins of the present compositions are polypeptides that produce poresor channels allowing passage of molecules across the outer membrane ofgram negative microbes (see, for instance, Nikaido and Vaara, OuterMembrane, In: Escherichia coli and Salmonella typhimurium, Cellular andMolecular Biology, Neidhardt et al., (eds.) American Society forMicrobiology, Washington, D.C., pp. 7–22 (1987)) and the membrane ofgram positive microbes. For instance, it is believed that the porinsproduced by gram negative microbes may include OmpA, OmpC, OmpD, OmpF,or PhoE. The porins are relatively conserved between gram negativebacteria, and play a role in iron binding. For example, OmpF and OmpCwill bind lactoferrin (Erdei et al., Infec. Immun., 62, 1236–1240(1994)), while OmpA will bind ferrichrome (Coulton et al., J. Gen.Microbiol., 110, 211–220 (1979)). Antibodies early in infectionparticularly of the IgM class have been found to cross-react with porinsof E. coli, Salmonella, Pasteurella, Pseudomonas and Klebsiella, andwill bind lactoferrin and/or ferrichrome, precluding the availability ofan iron source for microbial growth. Without intending to be limited bytheory, antibodies to these polypeptides will also bind to the porins onthe surface to enhance opsonization and/or complement-mediated bacteriallysis.

A composition of the present invention may contain at least two porinsisolated from one or more genera or one or more species of microbe. Insome aspects of the present invention, preferably the porins of acomposition are derived from multiple species of microbes of the samegenus of microbe, or from multiple strains of the same species ofmicrobe. In some aspects of the present invention, preferably the porinsof a composition are derived from the same microbe from which the SRPsof the composition were isolated.

Preferably, porins of the compositions of the present invention haveimmunogenic activity. Without intending to be limiting, porins of thepresent composition act as an adjuvant to enhance the immune response ofan animal to porins and SRPs present in a composition of the presentinvention when administered to an animal as described herein.

Preferably, the molecular weights of porins of the compositions of thepresent invention, as determined by separation of the porins using anabout 12% SDS-PAGE gel under reducing and denaturing conditions, arebetween about 30 kDa and about 43 kDa, more preferably, between about 33kDa and about 40 kDa. Preferably, the porins are obtained from a gramnegative microbe. Typically, different species of Salmonella eachproduce at least two porins. Preferably, when the composition includesporins from a Salmonella, the porins are isolated from one species ofSalmonella. Preferably, the molecular weights of porins isolated fromSalmonella spp. are between about 37 kDa to about 40 kDa, morepreferably, between about 38 kDa and about 39 kDa. Typically, E. coliproduces at least two porins. Preferably, the molecular weights ofporins isolated from E. coli are between about 33 kDa to about 39 kDa,more preferably, between about 34 kDa and about 38 kDa.

Preferably, porins of the present compositions can be identified usingantibodies that specifically bind porins. Such antibodies can be madeusing the E. coli strain having the designation ATCC #55652. Typically,ATCC #55652 is grown under low iron conditions, and porins are isolatedfrom the strain as described in Example 1, or as described by Emery etal. (U.S. Pat. No. 5,830,479). Antibody is then made that specificallybinds the porins. Laboratory methods for producing polyclonal andmonoclonal antibodies are routine and known in the art (see, forinstance, Harlow E. et al. Antibodies: A laboratory manual Cold SpringHarbor Laboratory Press, Cold Spring Harbor (1988) and Ausubel, R. M.,ed. Current Protocols in Molecular Biology (1994)). Methods fordetermining whether porins of the present compositions are specificallybound by antibodies made using porins isolated from ATCC #55652 areroutine and known to the art, and include, for instance, westernimmunoblot and enzyme linked immunosorbant assay.

Preferably, the compositions of the present invention include lowconcentrations, more preferably, undetectable concentrations, oflipopolysaccharide (LPS). LPS is a component of the outer membrane ofmost gram negative microbes (see, for instance, Nikaido and Vaara, OuterMembrane, In: Eseherichia coli and Salmonella typhimurium, Cellular andMolecular Biology, Neidhardt et al., (eds.) American Society forMicrobiology, Washington, D.C., pp. 7–22 (1987), and typically includespolysaccharides (0-specific chain, the outer and inner core) and thelipid A region. The lipid A component of LPS is the most biologicallyactive component of the LPS structure and together induce a widespectrum of pathophysiological effects in mammals. The most dramaticeffects are fever, disseminated intravascular coagulation, complementactivation, hypotensive shock, and death. LPS plays a major role in theactivation of various cell types, particularly those of lymphoid origin.This activation results in the production of an impressive array ofendogenous mediators that, in turn, activate the complement system,impair mitochondrial function, activate lysosomal activity, stimulateprostaglandin activity, and cause macrophage cytotoxicity andtumoricidal activity. This non-specific immunostimulatory activity ofLPS can enhance the formation of a granuloma at the site ofadministration of compositions that include LPS. Such reactions canresult in undue stress on the animal by which the animal may back offfeed or water for a period of time, and exasperate infectious conditionsin the animal. In addition, the formation of a granuloma at the site ofinjection can increase the likelihood of possible down grading of thecarcass due to scaring or blemishes of the tissue at the injection site(see, for instance, Rae, Injection Site Reactions, available on theworld wide web at animal.ufl.edu/short94/rae.htm

The concentration of LPS can be determined using routine methods knownto the art. Such methods typically include measurement of dye binding byLPS (see, for instance, Keler and Nowotny, Analyt. Biochem., 156, 189(1986)) or the use of a Limulus amebocyte lysate (LAL) test (see, forinstance, Endotoxins and Their Detection With the Limulus AmebocyteLystate Test, Alan R. Liss, Inc., 150 Fifth Avenue, New York, N.Y.(1982)). There are four basic commercially available methods that aretypically used with an LAL test: the gel-clot test; the turbidimetric(spectrophotometric) test; the colorimetric test; and the chromogenictest. An example of a gel-clot assay is available under the tradenameE-TOXATE (Sigma Chemical Co., St. Louis, Mo.; see Sigma TechnicalBulletin No. 210). Typically, assay conditions include contacting thecomposition with a preparation containing a lysate of the circulatingamebocytes of the horseshoe crab, Limulus polyphemus. When exposed toLPS, the lysate increases in opacity as well as viscosity and may gel.About 0.1 milliliter of the composition is added to lysate. Typically,the pH of the composition is between 6 and 8, preferably, between 6.8and 7.5. The mixture of composition and lysate is incubated for about 1hour undisturbed at about 37° C. After incubation, the mixture isobserved to determine if there was gelation of the mixture. Gelationindicates the presence of endotoxin. To determine the amount ofendotoxin present in the composition, dilutions of a standardizedsolution of endotoxin are made and tested at the same time that thecomposition is tested. Standardized solutions of endotoxin arecommercially available from, for instance, Sigma Chemical (Catalog No.210-SE) and U.S. Pharmacopeia (Rockville, Md., Catalog No. 235503). Inincreasing order of preference, a composition of the present inventionhas no greater than about 10.0 endotoxin units per milliliter (EU/ml),no greater than about 5.0 EU/ml, no greater than about 1.0 EU/ml, nogreater than about 0.5 EU/ml, no greater than about 0.2 EU/ml, nogreater than about 0.1 EU/ml, most preferably, no greater than about0.05 EU/ml. An endotoxin unit (EU) is defined in comparison to thecurrent FDA Endotoxin Reference Standard Lot EC-5. One vial of lot EC-5contains 10,000 EU. In general, about 1 nanogram (ng) of pure LPS isequal to between about 5 and about 10 endotoxin units.

The compositions of the present invention optionally further include apharmaceutically acceptable carrier. “Pharmaceutically acceptable”refers to a diluent, carrier, excipient, salt, etc, that is compatiblewith the other ingredients of the composition, and not deleterious tothe recipient thereof. Typically, the composition includes apharmaceutically acceptable carrier when the composition is used asdescribed below in “Methods of Use.” The compositions of the presentinvention may be formulated in pharmaceutical preparations in a varietyof forms adapted to the chosen route of administration, preferably,routes suitable for stimulating an immune response to an antigen. Thus,a composition of the present invention can be administered via knownroutes including, for example, oral; parental including intradermal,subcutaneous, intramuscular, intravenous, intraperitoneal, etc., andtopically, such as, intranasal, intrapulmonary, intramammary,intravaginal, intrauterine, etc. It is foreseen that a composition canbe administered to a mucosal surface, such as by administration to thenasal or respiratory mucosa (e.g. spray or aerosol), to stimulatemucosal immunity, such as production of secretory IgA antibodies,throughout the animal's body.

A composition of the present invention can also be administered via asustained or delayed release implant. Suitable implants are known. Someexamples of implants suitable for use according to the invention aredisclosed in Emery and Straub (WO 01/37810). Implants can be produced atsizes small enough to be administered by aerosol or spray. Implants alsoinclude nanospheres and microspheres.

A composition of the present invention is administered in an amountsufficient to provide an immunological response to SRPs and/or porinspresent in the composition, and/or increase performance characteristics.Performance characteristics are described in greater detail herein. Theamount of the polypeptide present in a composition of the presentinvention can vary. For instance, the dosage of polypeptide can bebetween about 0.01 micrograms (μg) and about 300 milligrams (mg),typically between about 0.1 mg and about 10 mg. For an injectablecomposition (e.g. subcutaneous, intramuscular, etc.) the polypeptide ispreferably present in the composition in an amount such that the totalvolume of the composition administered is about 0.5 ml to 5.0 ml,typically about 1.0–2.0 ml. The amount administered will vary dependingon various factors including, but not limited to, the specificpolypeptides chosen, the weight, physical condition and age of theanimal, and the route of administration. Thus, the absolute weight ofthe polypeptide included in a given unit dosage form can vary widely,and depends upon factors such as the species, age, weight and physicalcondition of the animal, as well as the method of administration. Suchfactors can be determined by one of skill in the art. Other examples ofdosages suitable for the invention are disclosed in Emery et al. (U.S.Pat. No. 6,027,736).

The formulations may be conveniently presented in unit dosage form andmay be prepared by methods well known in the art of pharmacy. Allmethods of preparing a composition including a pharmaceuticallyacceptable carrier include the step of bringing the active compound(e.g., SRPs and/or porins as described herein) into association with acarrier that constitutes one or more accessory ingredients. In general,the formulations are prepared by uniformly and intimately bringing theactive compound into association with a liquid carrier, a finely dividedsolid carrier, or both, and then, if necessary, shaping the product intothe desired formulations.

A composition including a pharmaceutically acceptable carrier can alsoinclude an adjuvant. An “adjuvant” refers to an agent that can act in anonspecific manner to enhance an immune response to a particularantigen, thus potentially reducing the quantity of antigen necessary inany given immunizing composition, and/or the frequency of injectionnecessary in order to generate an adequate immune response to theantigen of interest. Adjuvants may include for example, IL-1, IL-2,emulsifiers, muramyl dipeptides, dimethyldiocradecylammonium bromide(DDA), avridine, aluminum hydroxide, oils, saponins, alpha-tocopherol,polysaccharides, emulsified paraffins (available from under thetradename EMULSIGEN from MVP Laboratories, Ralston, Nebr.), ISA-70, RIBIand other substances known in the art.

In another embodiment, an composition of the invention including apharmaceutically acceptable carrier can include a biological responsemodifier, such as, for example, IL-2, IL-4 and/or IL-6, TNF, IFN-alpha,IFN-gamma, and other cytokines that effect immune cells. An immunizingcomposition can also include an antibiotic, preservative, anti-oxidant,chelating agent, etc. Such components are known in the art.

Another aspect of the present invention provides improved methods forobtaining SRPs from gram negative microbes and improved methods forobtaining porin polypeptides from gram negative microbes. The methodsinclude providing a gram negative microbe, disrupting the microbe,solubilizing the microbe, and isolating the polypeptides.

A gram negative microbe to be provided in the method is incubated underconditions that promote the expression of SRPs. Typically, suchconditions are low iron conditions. As used herein, the phrase “low ironconditions” refers to an environment, typically bacteriological media,that contains amounts of free iron that cause a microbe to express SRPs.As used herein, the phrase “high iron conditions” refers to anenvironment that contains amounts of free iron that cause a microbe tonot express SRPs. Preferably, low iron conditions are the result of theaddition of an iron chelating compound to media, and high ironconditions are present when a chelator is not present in the media.Examples of iron chelators include 2,2′-dipyridyl (also referred to inthe art as α,α′-bipyridyl), 8-hydroxyquinoline,ethylenediamine-di-O-hydroxyphenylacetic acid (EDDHA), desferrioxaminemethanesulphonate (desferol), transferrin, lactoferrin, ovotransferrin,biological siderophores, such as, the catecholates and hydroxamates, andcitrate. Preferably, 2,2′-dipyridyl is used. Typically, 2,2′-dipyridylis added to the media at a concentration of about 25micrograms/milliliter (μg/ml), more preferably, at about 50 μg/ml, mostpreferably, at about 100 μg/ml. The media used to incubate the microbeis not critical, and varies depending on the microbe. For instance, whenthe microbe is Salmonella spp. or E. coli, tryptic soy broth or brainheart infusion may be used. The volume of media used to incubate themicrobe can vary. When a microbe is being evaluated for the ability toproduce SRPs and porins, the microbe can be grown in a suitable volume,for instance, 10 milliliters to 1 liter of medium. When a microbe isbeing grown to obtain SRPs and porins for use in, for instance,administration to animals, the microbe may be grown in a fermentor toallow the isolation of larger amounts of polypeptides. Methods forgrowing microbes in a fermentor are routine and known to the art. Theconditions used for growing a microbe preferably include an ironchelator, preferably 2,2′-dipyridyl, a pH of between about 6.5 and about7.5, preferably between about 6.9 and 7.1, and a temperature of about37° C. Optionally, when a fermentor is used, dissolved oxygen ismaintained at between about 20% and about 40%, preferably, about 30%,but may vary depending on the metabolic requirements of the organism.

After growth, the gram negative microbe that is to be provided in themethod is harvested. Harvesting includes concentrating the microbe intoa smaller volume and suspending in a media different than the growthmedia. Methods for concentrating a microbe are routine and known to theart, and include, for example, centrifugation. Typically, theconcentrated microbe is suspended in decreasing amounts of buffer.Preferably, the final buffer includes a metal chelator, preferably,ethylenediaminetetraacetic acid (EDTA), which also aids in the releaseof lipopolysaccharide from the cell wall. Preferably, the final bufferalso minimizes proteolytic degradation. This can be accomplished byhaving the final buffer at a pH of greater than about 8.0, preferably,at least about 8.5, and/or including one or more proteinase inhibitors(e.g., phenylmethanesulfonyl fluoride). Optionally and preferably, theconcentrated microbe is frozen at −20° C. or below until disrupted.

The gram negative microbe may be disrupted using chemical, physical, ormechanical methods routine and known to the art, including, for example,french press, sonication, or homoginization. Preferably, homoginizationis used. As used herein, “disruption” refers to the breaking up of thecell. Disruption of a microbe can be measured by methods that areroutine and known to the art, including, for instance, changes inoptical density. Typically, a microbe is subjected to disruption untilthe optical density does not change after further disruption. Forinstance, if percent transmittance is measured, the microbe is disrupteduntil the percent transmittance does not increase after furtherdisruption. Preferably, the microbe is present in a buffer thatminimizes proteolytic degradation. Preferably, the microbe is present inthe buffer at a concentration of between about 720 grams of microbe per1,000 milliliters of buffer and about 1,080 grams of microbe per 1,000milliliters of buffer, more preferably, between about 810 grams ofmicrobe per 1,000 milliliters of buffer to about 990 grams of microbeper 1,000 milliliters of buffer, most preferably, about 900 gramsmicrobe per 1,000 milliliters of buffer. The temperature duringdisruption is typically kept low, preferably at about 4° C., to furtherminimize proteolytic degradation.

The disrupted microbe is solubilized in a detergent, for instance, ananionic, zwitterionic, nonionic, or cationic detergent. Preferably, thedetergent is sarcosine, more preferably, sodium lauroyl sarcosinate. Asused herein, the term “solubilize” refers to dissolving cellularmaterials (e.g., polypeptides, nucleic acids, carbohydrates) into theaqueous phase of the buffer in which the microbe was disrupted, and theformation of aggregates of insoluble cellular materials. The conditionsfor solubilization preferably result in the aggregation of SRPs and/orporins into insoluble aggregates that are large enough to allow easyisolation by, for instance, centrifugation. The ability to produceinsoluble aggregates was unexpected, and provides for an economical wayto isolate SRPs and porins.

Preferably, the sarcosine is added such that the final ratio ofsarcosine to gram weight of disrupted microbe is between about 0.8 gramsarcosine per about 4.5 grams pellet mass and about 1.2 grams sarcosineper about 4.5 grams pellet mass, preferably, about 1.0 gram sarcosineper about 4.5 grams pellet mass. The solubilization of the microbe maybe measured by methods that are routine and known to the art, including,for instance, changes in optical density. Typically, a disrupted microbeis allowed to solubilize until the percent transmitance at about 540 nmis between about 25% and about 30%. Preferably, the solubilization isallowed to occur for at least about 24 hours, more preferably, at leastabout 48 hours, most preferably, at least about 60 hours. Thetemperature during disruption is typically kept low, preferably at about4° C.

The insoluble aggregates that include the SRPs and porins may beisolated by methods that are routine and known to the art. Preferably,the insoluble aggregates are isolated by centrifugation. Typically,centrifugation of outer membrane polypeptides that are insoluble indetergents requires centrifugal forces of at least 50,000× g, typicallyabout 100,000× g. The use of such centrifugal forces requires the use ofultracentrifuges, and scale-up to process large volumes of sample isoften difficult and not economical with these types of centrifuges.Surprisingly and unexpectedly, the methods described herein provide forthe production of insoluble aggregates large enough to allow the use ofsignificantly lower centrifugal forces (for instance, about 46,000× g).Methods for processing large volumes at these lower centrifugal forcesare available and known to the art. Thus, the insoluble aggregates canbe isolated at a significantly lower cost.

Optionally and preferably, the sarcosine is removed from the isolatedSRPs and porins. Methods for removing sarcosine from the isolatedpolypeptides are known to the art, and include, for instance,diafiltration, precipitation, hydrophobic, ion-exchange, and/or affinitychromatography, and ultra filtration and washing the polypeptides inalcohol by diafiltration. After isolation, the polypeptides suspended inbuffer and stored at low temperature, for instance, −20° C. or below.

Another unexpected observation was that this method for obtaining SRPsand proins from a gram negative microbe also resulted in SRPs and porinscontaining low amounts of LPS. LPS is a potent immunostimulant, and whenpresent in compositions that are administered to animals, especiallymammals, can result in decreases in certain performance characteristics,and/or injection site reactions that can result in the downgrading ofcarcasses due to scaring or blemishes of tissue at the injection site.The ability to isolate SRPs and porins with low amounts of LPS resultsin decreased economic losses associated with administration ofpreparations from gram negative microbes. The decreased amount of LPSresults in fewer condemned and/or downgraded carcasses at slaughter, andfewer decreases in performance characteristics.

SRPs may also be isolated from gram positive microbes using methods thatare known to the art. The isolation of SRPs from gram positive microbescan be accomplished as described in, for instance, Hussain, et al.Infect. Immun., 67, 6688–6690 (1999); Trivier, et al., FEMS Microbiol.Lett., 127, 195–199 (1995); Heinrichs, et al., J. Bacteriol., 181,1436–1443 (1999).

Methods of Use

An aspect of the present invention is further directed to methods ofusing the compositions of the present invention. The methods includeadministering to an animal an effective amount of a composition of thepresent invention. Preferably, the composition includes LPS at aconcentration of, in increasing order of preference, no greater thanabout 10.0 endotoxin units per milliliter (EU/ml), no greater than about5.0 EU/ml, no greater than about 1.0 EU/ml, no greater than about 0.5EU/ml, no greater than about 0.1 EU/ml, most preferably, no greater thanabout 0.05 EU/ml. Preferably, the composition further includes apharmaceutically acceptable carrier. The animal can be, for instance,avian (including, for instance, chickens or turkeys), bovine (including,for instance, cattle), caprine (including, for instance, goats), ovine(including, for instance, sheep), porcine (including, for instance,swine), Bison (including, for instance, buffalo), companion animals(including, for instance, horses), members of the family Cervidae(including, for instance, deer, elk, moose, caribou and reindeer), andhumans.

In some aspects, the methods may further include additionaladministrations (e.g., one or more booster administrations) of thecomposition to the animal to enhance or stimulate a secondary immuneresponse. A booster can be administered at about 1 week to about 8weeks, preferably about 2 to about 4 weeks, after the firstadministration of the composition. Subsequent boosters can beadministered one, two, three, four, or more times annually. Withoutintending to be limited by theory, it is expected that annual boosterswill not be necessary, as an animal will be challenged in the field byexposure to microbes expressing SRPs and/or porins having epitopes thatare identical to or structurally related to epitopes present on the SRPsand/or porins of the composition administered to the animal.

In one aspect, the invention is directed to methods for inducing theproduction of antibody in an animal. The antibody produced includesantibody that specifically binds at least one polypeptide (an SRP and/ora porin) present in the composition. In this aspect of the invention, an“effective amount” is an amount effective to result in the production ofantibody in the animal. Methods for determining whether an animal hasproduced antibodies that specifically bind polypeptides present in acomposition of the present invention can be determined as describedherein.

The method may be used to produce antibody that specifically bindspolypeptides, preferably, SRPs and/or porins, present on the surface ofa microbe other than the microbe from which the SRPs and porins of thecomposition were isolated. As discussed herein, SRPs and porinstypically include epitopes that are conserved in the SRPs and porins ofdifferent species and different genera of microbes. Accordingly,antibody produced using SRPs and porins from one microbe are expected tobind to SRPs and/or porins present on other microbes (see, for instance,Examples 8 and 10) and provide broad spectrum protection against grampositive and gram negative organisms. Examples of gram positive microbesto which the antibody specifically binds are members of the familyMicrococcaceae, members of the family Deinococcaceae, or other grampositive microbes as described in the section “Compositions.”Preferably, gram positive microbes to which the antibody binds areStaphylococcus aureus, Streptococcus agalactiae, Streptococcus uberis,Streptococcus bovis and Streptococcus dysgalatiae, Streptococcuszooepidemicus, and Streptococcus equi, most preferably, Staphylococcusaureus. Examples of gram negative microbes to which the antibodyspecifically binds are enteropathogens, more preferably, members of thefamily Enterobacteriaceae, even more preferably, members of theEnterobacteriaceae tribes Escherichieae or Salmonelleae, as described inthe section “Compositions.” Most preferably, gram negative microbes towhich the antibody specifically binds are Salmonella spp. and E. coli.

In an alternative aspect, methods for inducing the production ofantibody in an animal include administering a composition prepared froma whole cell preparation. According to this embodiment, the whole cellpreparation can be prepared from, for example, a modified Escherichiacoli such as a virulent R-mutant, as for example, E. coli J5(commercially available from ATCC as ATCC #43745; described by Overbecket al., J. Clin. Microbiol., 25, 1009–1013 (1987)), or SalmonellaMinnesota (commercially available from ATCC as ATCC number #49284; asdescribed by Sanderson et al., J. Bacteriol., 119, 753–759, 760–764(1974)) that lack outer oligosaccharide side chains of LPS. In anon-immunized animal outer oligosaccharide side chains tend to mask SRPson the cell membrane in such a way that the immune system does notrecognize the SRPs and production of anti-SRP antibody titers aredepressed. Thus, to enhance the immune stimulating capability of animmunizing composition made with intact bacterial cells to elicit ananti-SRP immune response, the cell membrane can be chemically altered toeliminate the interfering oligosaccharide side chains or a mutantorganism such as the E. coli J5 organism discussed above can be used.Chemically modified cells or mutants are then grown underiron-restriction conditions to enhance SRP production as described in,for example, U.S. Pat. No. 6,027,736.

In another aspect, the present invention is directed to methods fortreating certain conditions in animals that may be caused by, orassociated with, a microbe. Such conditions include, for instance, gramnegative microbial infections and gram positive microbial infections.Examples of conditions caused by microbial infections include mastitis,fecal shedding of a microbe, metritis, strangles, intrauterineinfections, odema disease, enteritis, chronic reproductive infections,laminitis, and acute or chronic Chlamydiosis, Colibacillosis,Ehrlichiosis, Leptospirosis, Pasteurellosis, Pseudotuberculosis,Salmonellosis. Examples of conditions that may be caused by microbialinfections include performance characteristics such as decreased milkproduction, high somatic cell counts, and weight loss. Treatment ofthese conditions can be prophylactic or, alternatively, can be initiatedafter the development of a condition described herein. Treatment that isprophylactic, for instance, initiated before a subject manifestssymptoms of a condition caused by a microbe, is referred to herein astreatment of a subject that is “at risk” of developing the condition.Typically, an animal “at risk” of developing a condition is an animalpresent in an area where the condition has been diagnosed and/or islikely to be exposed to a microbe causing the condition. Accordingly,administration of a composition can be performed before, during, orafter the occurrence of the conditions described herein. Treatmentinitiated after the development of a condition may result in decreasingthe severity of the symptoms of one of the conditions, or completelyremoving the symptoms. Preferably, administration of a compound isperformed before the occurrence of the conditions described herein. Inthis aspect of the invention, an “effective amount” is an amounteffective to prevent the manifestation of symptoms of a disease,decrease the severity of the symptoms of a disease, and/or completelyremove the symptoms. The potency of a composition of the presentinvention can be tested according to standard methods established by 9CFR § 113. For instance, 9 CFR § 113.120(c) and 9 CFR § 113.123(c)describe standard methods for determining the potency of the compositionagainst a standard reference bacterin of Salmonella typhimurium andSalmonella dublin, respectively. Methods for determining whether ananimal has the conditions disclosed herein and symptoms associated withthe conditions are routine and known to the art.

In one aspect the invention is also directed to treating a gram negativemicrobial infection in an animal, and/or a gram positive infection in ananimal. The method includes administering an effective amount of thecomposition of the present invention to an animal having or at risk ofhaving a gram positive or a gram negative infection, and determiningwhether at least one symptom of infection is reduced.

In another aspect, the invention provides for treatment of mastitis inmilk producing animals, such as cattle. The method includesadministering an effective amount of the composition of the presentinvention to a milk producing animal having or at risk of havingmastitis, and determining whether at least one symptom of mastitis isreduced. Mastitis refers to inflammation of the mammary gland. Physical,chemical and usually bacteriological changes in the milk andpathological changes in the glandular tissue characterize it. Theseglandular changes often result in a number of symptomatic conditionssuch as, discoloration of the milk, the presence of clots and thepresence of large numbers of leukocytes. Clinically, mastitis is seen asswelling, heat, pain and induration in the mammary gland often resultingin deformation of the udder. In many cases the diagnosis of subclinicalinfections has come to depend largely on indirect tests which depend onthe leukocyte content of the milk or somatic cell count (SCC). The mostcommon organisms that infect the udder are classified into twogroups: 1) contagious pathogens and 2) environmental pathogens. Examplesof contagious pathogens include, for instance, Staphylococcus aureus andStreptococcus agalactiae. Examples of environmental pathogens includethe coliforms such as, Escherichia coli, Klebsiella pneumoniae,Klebsiella oxytoca, Enterococcus faecium, Enterococcus faecalis,Enterobacter aerogenes, and Streptococci such as S. uberis, S. bovis andS. dysgalactiae. Examples of other gram negative bacteria which maycause mastitis include, Aerobacter spp., Bacteroides spp., Campylobacterspp., Citrobacter spp., Enterobacter spp., Erwinia spp., Escherichiaspp., Fusobacaterium spp., Klebsiella spp., Leptospira spp., Mycoplasmaspp., Pasteurella spp., Providencia spp., Pseudomonas spp., Proteusspp., Serratia spp., Salmonella spp., and Yersinia spp. Preferably,administration of the composition of the present invention will treatmastitis caused by a gram negative microbe or a gram positive microbe.Preferably, mastitis-causing gram positive microbes that can be treatedusing the present invention are members of the family Micrococcaceae,members of the family Deinococcaceae, or other gram positive microbes asdescribed in the section “Compositions.” More preferably, gram positivemicrobes are Staphylococcus aureus, Streptococcus agalactiae,Streptococcus uberis, Streptococcus bovis and Streptococcus dysgalatiaeand Streptococcus equi, most preferably, Staphylococcus aureus.Preferably, mastitis-causing gram negative microbes that can be treatedusing the present invention are enteropathogens, more preferably,members of the family Enterobacteriaceae, even more preferably, membersof the Enterobacteriaceae tribes Escherichieae or Salmonelleae, asdescribed in the section “Compositions.” Most preferably, gram negativemicrobes are Salmonella spp. and E. coli.

In yet another aspect, the invention provides for treatment of metritisin an animal, preferably in cattle. The method includes administering aneffective amount of the composition of the present invention to ananimal having or at risk of having metritis, and determining whether atleast one symptom of metritis is reduced. Metritis is an inflammation ofthe uterus after calving and is often caused by a retained placenta.Subclinical metritis in an animal is often indicative of decreasedperformance characteristics, including, for instance, lower milkproduction, decreased fertility and weight loss, of the animal.

In another aspect, the invention is directed to a method for treatinghigh somatic cell counts in an animal's milk, preferably, a cow. Themethod includes administering an effective amount of the composition ofthe present invention to a milk producing animal having or at risk ofhaving high somatic cell counts, and determining whether the somaticcell count in milk obtained from the animal contains reduced somaticcell counts compared to milk obtained from the animal before receivingthe composition. In another aspect the invention is directed to a methodfor reducing somatic cell counts in an animal's milk. Surprisingly andunexpectedly, decreases in somatic cell counts in animals receiving SRPsand porins from Salmonella did not appear to be related to clinicaldisease caused by Salmonella (see results section of Example 7, andExample 8). Somatic cell count (SCC) is a commonly used measure of milkquality. Somatic cells include leucocytes of the animal, and aretypically present at low levels in normal milk. High levels of somaticcells in milk, for instance, at least about 250,000 cells per milliliterof milk, preferably, at least about 400,000 cells per milliliter ofmilk, indicate reduced milk quality. High levels of somatic cells inmilk may be indicative of infection (mastitis), but may also beunassociated with infection (see Example 8). SCC is monitored, typicallyby milk processing plants, using methods that are routine to the art. Inone aspect, the invention is particularly advantageous for reducingsomatic cell counts of milk produced by milk producing animals infectedwith a microbe from the families Acholeplasmataceae, Bacteroidaceae,Enterobacteriaceae, Leptospiraceae, Micrococcaceae, Mycoplasmataceae,Mycobacteriaceae, Neisseriaceae, Pasteurellaceae, Pseudomonadaceae,Spirochaetaceae, or Vibronaceae. Preferably, the SCC is reduced to, inincreasing order of preference, less than about 750,000 cells/ml, lessthan about 600,000 cells/ml, less than about 400,000 cells/ml, mostpreferably, less than about 250,000 cells/ml. Gram positive microbescausing increased SCC that can be treated using the present method aremembers of the family Micrococcaceae, members of the familyDeinococcaceae, or other gram positive microbes as described in thesection entitled “Compositions.” Preferably, gram positive microbes areStaphylococcus aureus, Streptococcus agalactiae, Streptococcus uberis,Streptococcus bovis and Streptococcus dysgalatiae and Streptococcusequi, most preferably, Staphylococcus aureus. Gram negative microbescausing increased SCC that can be treated using the present method areenteropathogens, more preferably, members of the familyEnterobacteriaceae, even more preferably, members of theEnterobacteriaceae tribes Escherichieae or Salmonelleae, as described inthe section entitled “Compositions.” Most preferably, gram negativemicrobes to which the antibody specifically binds are Salmonella spp.and E. coli.

In another aspect, the invention is directed to treating low milkproduction by a milk producing animal, preferably, a cow. The methodincludes administering an effective amount of the composition of thepresent invention to a milk producing animal having or at risk of havinga low milk production, and determining whether milk production by theanimal is increased compared to milk production by the animal beforereceiving the composition. In another aspect the invention is directedto a method for increasing milk production in a milk producing animal,preferably, a cow. The method includes administering a composition ofthe present invention to a milk producing animal, and determiningwhether milk production by the animal is increased compared to milkproduction by the animal before receiving the composition. Preferably,the milk production by a milk producing animal after administration ofcomposition of the present invention is increased by at least about 1%,more preferably, by at least about 3%, most preferably, by at leastabout 6%. Preferably, milk production by a cow is determined beforeadministration and about 2 weeks, more preferably, about 8 weeks, mostpreferably, about 16 weeks after administration of the composition.

In yet another aspect, the invention is directed to treating intestinalcolonization by a microbe, preferably, an enteropathogen. Intestinalcolonization by an enteropathogen is typically determined by measuringfecal shedding of a microbe by the animal. The method for treatingintestinal colonization by an enteropathogen includes administering aneffective amount of the composition of the present invention to ananimal having or at risk of having fecal shedding of an enteropathogen,and determining whether the fecal shedding of an enteropathogen isdecreased compared to the fecal shedding of the microbe by the animalbefore receiving the composition. Fecal shedding may be measured bymethods routine and known to the art. Many of the animals infected withan enteropathogen, for instance, Salmonella spp. or E. coli, will shedthe microbe in their feces or body excretions. When the microbe isSalmonella, this may serve as a source for chronic Salmonellosis in theherd. Preferably, the microbe is E. coli or a Salmonella spp., morepreferably, a Salmonella spp. Preferably, the microbe includes apolypeptide (for instance, an SRP and/or a porin) that include anepitope that is structurally related to an epitope present on an SRPand/or a porin present in the composition administered to the animal.Preferably, the level of fecal shedding is reduced by about 10-fold,more preferably, by about 100-fold, even more preferably, by about1,000-fold. Most preferably, the level of fecal shedding of anenteropathogen is reduced such that the enteropathogen is no longerdetectable.

The present invention is also directed to methods of increasing milkquality. Indicators of low milk quality include, for instance, somaticcell counts of at least about 250,000 cells per milliliter of milk,preferably, at least about 400,000 cells per milliliter of milk, andmicrobial contamination of milk. The method includes administering aneffective amount of the composition of the present invention to ananimal, and determining whether the quality of milk from a milkproducing animal is increased compared to the milk quality of the milkproducing animal before receiving the composition. Without intending tobe limited by theory, milk produced by these animal results in thepresence of antibody directed to SRPs and porins in the milk, and theseantibodies will decrease the ability of microbes having cross-reactiveSRPs and/or cross-reactive porins to grow in the milk.

A composition of the invention can be used to provide for active orpassive immunization against bacterial infection. Generally, thecomposition can be administered to an animal to provide activeimmunization. However, the composition can also be used to induceproduction of immune products, such as antibodies, which can becollected from the producing animal and administered to another animalto provide passive immunity. Immune components, such as antibodies, canbe collected to prepare antibody compositions from serum, plasma, blood,colostrum, etc. for passive immunization therapies. Antibodycompositions comprising monoclonal antibodies and/or anti-idiotypes canalso be prepared using known methods. Passive antibody compositions andfragments thereof, e.g., scFv, Fab, F(ab′)₂ or Fv or other modifiedforms thereof, may be administered to a recipient in the form of serum,plasma, blood, colostrum, and the like. However, the antibodies may alsobe isolated from serum, plasma, blood, colostrum, and the like, usingknown methods and spray dried or lyophilized for later use in aconcentrated or reconstituted form. Passive immunizing preparations maybe particularly advantageous for treatment of acute systemic illness, orpassive immunization of young animals that failed to receive adequatelevels of passive immunity through maternal colostrum.

Another aspect of the present invention provides methods for detectingantibody that specifically binds polypeptides of the compositions of thepresent invention. These methods are useful in, for instance, detectingwhether an animal has antibody that specifically bind polypeptides ofthe compositions of the present invention, and diagnosing whether ananimal may have a condition caused by a microbe expressing SRPs and/orporins of the compositions described herein. Preferably, such diagnosticsystems are in kit form. The methods include contacting an antibody witha preparation that includes polypeptides present in a composition of thepresent invention to result in a mixture. Preferably, the antibody ispresent in a biological sample, more preferably blood, milk, orcolostrum. The method further includes incubating the mixture underconditions to allow the antibody to specifically bind the polypeptide toform a polypeptide:antibody complex. As used herein, the term“polypeptide:antibody complex” refers to the complex that results whenan antibody specifically binds to a polypeptide. The preparation thatincludes the polypeptides present in a composition of the presentinvention may also include reagents, for instance a buffer, that provideconditions appropriate for the formation of the polypeptide:antibodycomplex. The polypeptide:antibody complex is then detected. Thedetection of antibodies is known in the art and can include, forinstance, immunofluorescence and peroxidase.

The methods for detecting the presence of antibodies that specificallybind to polypeptides of the compositions of the present invention can beused in various formats that have been used to detect antibody,including radioimmunoassay and enzyme-linked immunosorbent assay.

The present invention also provides a kit for detecting antibody thatspecifically binds polypeptides of the compositions of the presentinvention. The kit includes at least two SRPs and at least two porins ina suitable packaging material in an amount sufficient for at least oneassay. Optionally, other reagents such as buffers and solutions neededto practice the invention are also included. Instructions for use of thepackaged polypeptides are also typically included.

As used herein, the phrase “packaging material” refers to one or morephysical structures used to house the contents of the kit. The packagingmaterial is constructed by wellknown methods, preferably to provide asterile, contaminant-free environment. The packaging material has alabel which indicates that the polypeptides can be used for detectingSRPs and/or porins. In addition, the packaging material containsinstructions indicating how the materials within the kit are employed todetect SRPs and porins. As used herein, the term “package” refers to asolid matrix or material such as glass, plastic, paper, foil, and thelike, capable of holding within fixed limits the polypeptides. Thus, forexample, a package can be a microtiter plate well to which microgramquantities of polypeptides have been affixed. “Instructions for use”typically include a tangible expression describing the reagentconcentration or at least one assay method parameter, such as therelative amounts of reagent and sample to be admixed, maintenance timeperiods for reagent/sample admixtures, temperature, buffer conditions,and the like.

The present invention is illustrated by the following examples. It is tobe understood that the particular examples, materials, amounts, andprocedures are to be interpreted broadly in accordance with the scopeand spirit of the invention as set forth herein.

EXAMPLES

Compositions including siderophore receptor proteins and porins fromSalmonella was evaluated for efficacy against a virulent challenge inmice and for the control of Salmonellosis in commercial dairy and feedlot cattle. The efficacy of the composition was evaluated by collectingdata on the following parameters: first the potency of the immunizingcomposition was evaluated against a live virulent challenge in mice, andsecondly the efficacy was evaluated in commercial dairy and feed lotcattle by examining the serological response to vaccination, eliminationof Salmonella as examined by fecal shedding, reduction in morbidity andmortality, reduction of somatic cells in milk, total milk production,and examination of injections sites after each vaccination.

Example 1 Production and Isolation of Siderophore Receptor Proteins andPorins

Gram negative bacteria belonging to the families Enterobacteriaceae andPseudomonadaceae, as well as other gram negative bacteria can be grownunder controlled fermentation conditions so as to express siderophorereceptor proteins and porins, and optionally, iron regulated proteins,on the outer membrane. The bacteria can be harvested by conventionalmethods and the outer membrane proteins can then be isolated and used asimmunogens in a vaccine composition described in detail in the followingexample.

Salmonella dublin was isolated from Holstein steers in a commercial feedlot showing clinical signs of Salmonellosis, and designated MS010207.The isolate was serotyped by the Minnesota Poultry Testing Laboratory,(Willmar, Minn.). A master seed stock of the organism was prepared byinoculating 100 ml of Tryptic Soy Broth (Difco Laboratories, Detroit,Mich.) containing 50 micrograms per milliliter (μg/ml) of 2,2-dipyridyl(Sigma-Aldrich St. Louis, Mo.). The culture was grown while stirring at200 rpm for 6 hours at 37° C. The bacteria were collected bycentrifugation at 10,000× g. The bacterial pellet was resuspended in 20ml physiological saline (0.85%) containing 20% glycerol. The bacterialsuspension was sterilely dispensed into 20–2 ml cryogenic vials andstored at −90° C. The master seed was expanded into a working seed thatwas then used for the production of siderophore receptor proteins andporins. A large-scale production process was developed involvingfermentation, bacterial harvest, disruption, solubilization,concentration, diafiltration, and isolation of final product.

Fermentation

A cryogenic vial of the working seed (1 ml at 10⁹ CFU/ml) was used toinoculate 500 ml of Tryptic Soy Broth (TSB) without dextrose (Difco)pre-warmed to 37° C. containing 50 micrograms 2,2-dipyridyl (Sigma), 2.7grams BiTek yeast extract (Difco) and glycerol (3% vol/vol). The culturewas incubated at 37° C. for 12 hours while stirring at 200 rpm at whichtime was inoculated into 2 liters of the above media and allowed to growfor an additional 4 hours at 37° C. This culture was used to inoculate a20-liter Virtis bench-top fermentor, (Virtis, Gardiner, N.Y.) chargedwith 13 liters of the above-described media. The pH was held constantbetween 6.9 and 7.1 by automatic titration with 30% NaOH and 10% HCL.The stirring speed was adjusted at 400 rev/minute, and the cultureaerated with 11 liters air/minute at 37° C. Foaming was controlledautomatically by the addition of 11 ml defoamer (Mazu DF 204 Chem/Serv,Minneapolis, Minn.). The culture was allowed to grow continuously atthese conditions for 4 hours at which time was sterilely pumped into a150-liter fermentor (W. B. Moore, Easton, Pa.). The fermentor wascharged with 115 liters tryptic soy broth without dextrose (3,750.0grams), BiTek yeast extract (625 grams), glycerol (3750 ml),2,2-dypyrdyl (3.13 grams) and Mazu DF 204 defoamer (100 ml). Theparameters of the fermentation were as follows: dissolved oxygen (DO)was maintained at 30% +/−10% by increasing agitation to 220 rev/minutesparged with 60 liters of air/minute and 10 pounds per square inch (psi)back pressure. The pH was held constant between 6.9 and 7.1 by automatictitration with 30% NaOH and 10% HCL. The temperature was maintained at37° C. At hour 4.5 (OD₅₄₀ 8–9) of the fermentation the culture wassupplemented with additional nutrients by feeding 7 liters of mediacontaining 1,875 grams TSB without dextrose, 313 grams yeast extract3.13 grams 2,2-dipyridyl and 1,875 ml of glycerol. The rate of feed wasadjusted to 29 ml/minute while increasing agitation to 675 rpm. At theend of the feed (hour 8.5) the fermentation was allowed to continue foran additional three hours at which point the fermentation was terminatedby lowing the temperature of the fermentor to 10° C. (OD₅₄₀ 35–40 at a1:100 dilution). The culture was sterilely transferred to a 200-litertank (LEE Process Systems and Equipment model 2000LDBT) in preparationfor harvest.

Harvest

The bacterial fermentation was concentrated and washed using a PallFiltron Tangential Flow Maxiset-25 (Pall Filtron Corporation, Northboro,Mass.) equipped with two 30 ft² Alpha 300-K open channel filters,catalog No. AS300C5, (Pall Filtron) connected to a Waukesha Model U-60feed pump (Waukesha Cherry-Burrell, Delevan, Wis.) The original culturevolume of 125 liters was reduced to 25 liters (2.5 liters/minute) usinga filter inlet pressure of 15 psi and a retentate pressure of 0 psi. Thebacterial retentate was adjusted back up to 50 liters usingphysiological saline (0.85%) and then concentrated again to 15 liters tohelp remove any contaminating exogenous proteins, etc. The retentate (15liters) was adjusted to 35 liters using sterile Osmotic Shock Buffer(OMS) containing 7.26 grams/liter Tris-base and 0.93 grams/liter EDTAadjusted to a pH of 8.5. The EDTA in the OMS serves to remove much ofLPS from the cell wall, while the elevated pH prevents much of theproteolytic degradation after freezing and disruption. Proteaseinhibitors may be used instead of, or in addition to, an elevated pH.The retentate was mixed thoroughly while in the 200-liter tank using abottom mount magnetically driven mixer. The retentate was sterilelydispensed (3.5 liters) into sterile 4 liter Nalgene containers No. 2122and placed into a −20° C. freezer for storage. Freezing the bacterialpellet serves to weaken the cell wall structure making downstreamdisruption more efficient. The pellet mass was calculated bycentrifuging 30 ml samples of the fermented culture and final harvest.Briefly, pre-weighted 50 ml Nalgene conical tubes were centrifuged at39,000×g for 90 minutes in a Beckman J2–21 centrifuge using a JA-21rotor (Beckman Instruments, Palo Alto Calif.). At the end of the run,the supernate was poured off and the tubes were weighed again. Thepellet mass was calculated for each stage. The fermentation processyielded a wet pellet mass of 9.0 kilograms.

Disruption (Homogenization)

Twenty kilograms of frozen bacterial cell slurry in OMS were thawed at4° C. (20 kg of pellet mass). The liquid culture suspension from eachcontainer was aseptically aspirated into a steam in place 250 literjacketed process tank (Lee, Model 259LU) with a top mounted mixer(Eastern, Model TME-½, EMI Incorporated, Clinton, Conn.) containing 222liters OMS pH 8.5 containing 0.1 grams thimerosal/liter as preservative.The volume of OMS was determined by dividing the pellet mass (in grams)by 900 and then multiplying the result by 10 to get the homogenizingvolume in liters (gram pellet mass/900×10=liters homogenizing volume).The bulk bacterial suspension was chilled to 4° C. with continuousmixing for 18 hours at 200 rpm at which time was disrupted byhomogenization. Briefly, the 250 liter tank containing the bacterialsuspension was connected to a model 12.51 H Rannie Homogenizer, (APVSystems, Rosemont, Ill.). A second 250 liter jacketed process tank(empty) was connected to the homogenizer such that the fluid in theprocess tank could be passed through the homogenizer, into the emptytank and back again, allowing for multiple homogenizing passes whilestill maintaining a closed system. The temperature during homogenizationwas kept at 4° C. At the start of each pass, fluid was circulated at 70psi via a Waukesha model 10DO pump (Waukesha) through the homogenizer(160 gallons/hour) and back to the tank of origin, while the homogenizerpressure was adjusted to 13,500 psi. Prior to the first pass, twopre-homogenizing samples were withdrawn from the homogenizer toestablish a baseline for determining the degree of disruption andmonitoring of pH. The degree of disruption was monitored bytransmittance (% T at 540 nm at 1:100 dilution) compared to thenon-homogenized sample. The number of passes through the homogenizer wasstandardized for different organisms based on the integrity of the cellwall and variation in the degree of disruption, which had a directcorrelation in the efficiency of solubilization and quality of endproduct. For example, the disruption of Salmonella passed three timesthrough the homogenizer gave a final percent transmittance between78–83% T at a 1:100 dilution. E. coli having the same pellet mass andstarting OD gave a % T of 86–91% (at a 1:100 dilution) after the thirdpass. It has been observed that bacteria differ in their cell wallintegrity and vary in their capacity of disruption under identicalcondition. This variation can effect the degree and efficiency ofsolubilization and recovery of SRPs and porins from the outer membrane.In general, cells were passed through the homoginizer until thetransmittance did not increase after an additional pass.

After homogenization, Sodium Lauroyl Sarcosinate (Hamptosyl L-30,Chem/Serv) was aseptically added to the homogenized bacterial suspensionfor solubilization. The amount of Sarcosine (30%) added equaled 0.0664times the solubilizing volume, in liters, (1.0 gram sarcosine/4.5 gramspellet mass). The tank was removed from the homogenizer and put onto achiller loop at 4° C. and mixed at 240 rpm for 60–70 hours. This timeperiod was important for complete solubilization. It was discovered thatincreasing the solubilization time in OMS at an elevated pH (8.0–8.5)that the SRPs and porins aggregated together forming large insolubleaggregates that were easily removed by centrifugation. The optimal ODafter solubilization was usually between 25–30% T at 540 nm.

Protein Harvest

The aggregated siderophore receptor proteins and porins within thesolubilized process fluid were collected by centrifugation using T-1Sharples, (Alfa Laval Seperations, Warminster, Pa.). Briefly, the tankof solubilized homogenate was fed into six Sharples with a feed rate of250 ml/minute at 17 psi at a centrifugal force of 46,000×g. The effluentwas collected into a second 250 liter jacketed process tank through aclosed sterile loop allowing for multiple passes through the centrifugeswhile maintaining a closed system. The temperature during centrifugationwas kept at 4° C. The solubilized homogenate was passed 8 times acrossthe centrifuges. Fifty percent of the protein was collected after thesecond pass, at which point, the solubilized fluid was concentrated to ⅓of its original volume, which shortened the process time for the next 6passes. Briefly, the solubilized homogenate tank was asepticallydisconnected from the centrifuges and connected to a Millipore PelliconTangential Flow Filter assembly (Millipore Corporation, Bedford, Mass.),equipped with a 25ft² screen-channel series Alpha 10K Centrasette filter(Pall Filtron) connected to a Waukesha Model U30 feed pump forconcentration. After concentration, centrifugation was continued untilthe process was completed. Protein was collected after each pass. Theprotein was collected, resuspended and dispensed in 50 litersTris-buffer pH 8.5 containing 0.3% formulin (Sigma) as preservative.

Diafiltration

The protein suspension was washed by diafiltration at 4° C. to removeany contaminating sarcosine that may be bound to the protein. Briefly,the 50 liters of protein was sterilely aspirated into a 200 literprocess tank containing 50 liters sterile Tris-buffer, pH 8.5 equippedwith a bottom mount Dayton mixer, Model 2Z846 (Dayton Electric, Chicago,Ill.) rotating at 125 rev/minute. The process tank was sterilelyconnected to a Millipore Pellicon Tangential Flow Filter assembly(Millipore Corporation), equipped with a 25ft² screen-channel seriesAlpha 10K Centrasette filter (Pall Filtron) connected to a WaukeshaModel U30 feed pump. The 100 liter protein solution was concentrated byfiltration to a target volume of 5.45 times the protein pellet mass atwhich point Tris-buffer pH 7.4 containing 5% isopropyl alcohol wasslowly added to the concentrate from a second process tank. Isopropylalcohol causes a slight unfolding of the protein structure allowing forthe removal of bound sarcosine without compromising the immunogenicityof the protein. Diafiltration continued until the pH stabilized to 7.4at which point 50 liters Tris-buffer pH 7.4 was slowly added bydiafiltration to remove residual alcohol. The protein suspension wasthen concentrated to approximately 25 liters. The protein concentratewas aseptically dispensed (3.5 liters) into sterile 4 liter Nalgenecontainers and placed into a −20° C. freezer for storage.

This process produces an extremely pure composition of SRPs and porinswith almost the complete removal of LPS with very little to no sarcosineresidue. The protein was examined by SDS-PAGE for purity and bandingprofile, bacterial contamination, residual sarcosine and LPS. Thebanding profile of the finished product showed consistent patterns asexamined by electrophoresis. The composition was tested for sarcosine bythe use of a modified agar gel diffusion test in which sheep red bloodcells (5%) were incorporated into an agar base (1.5%). Wells were cutinto the agar and samples of the finished product along with controlsamples of known concentrations of sarcosine at 0.05, 0.1, 0.2, 0.3,0.4, 0.5. 1.0 and 2.0% were placed into the wells. The gel was incubatedat 25° C. for 24 hours and the degree of hemolysis was determinedcompared to the controls. The process removes the level of detectablesarcosine below 0.05%, which at this concentration showed minimalhemolysis in control samples.

LPS was removed below the detection level as examined by a Limulusamebocyte lysate (LAL) test available under the tradename E-TOXATE(Sigma Chemical Co., St. Louis, Mo.).

Example 2 Mouse Vaccination and Challenge Study

The efficacy of a Salmonella dublin vaccine consisting of Siderophorereceptor proteins (SRPs) and porins was carried out against a livevirulent challenge in mice as described under 9 CFR 113.123. Sixtyfemale CF-1 mice obtained from Harlan Breeding Laboratories(Indianapolis, Ind.) weighing 16–22 grams were equally distributed into6 polycarbonate mouse cages (Ancore Corporation, Bellmore, N.Y.)designated as groups 1–6.

The composition including siderophore receptor proteins and porins wasprepared as described in Example 1 from a bovine field isolate ofSalmonella dublin originating from a herd of Holstein Dairy cows showingclinical symptoms of Salmonellosis.

The SRPs had molecular weights of 89 kDa, 84 kDa, 72 kDa and porins hadmolecular weights of 38–39 kDa as examined on a 12% SDS-Page gel. TheSRPs and porins in 8.3 ml (6,035 μg/ml) were resuspended into 69.2 mlphysiological saline (0.85%). The aqueous protein suspension (77.5 ml)was emulsified into 22.5 ml EMULSIGEN, (MVP Laboratories, Ralston,Nebr.) using a IKA Ultra Turrax T-50 homogenizing vessel (IKA,Cincinnati, Ohio) to give a final dose of 125 μg total protein in a 0.25ml injectable volume at a 22.5% vol/vol adjuvant concentration. Themouse dose was adjusted to be equivalent to a field dose of 1,000 μg ata 2 ml volume.

The potency of the vaccine was tested at four different concentrations,non-diluted (Group-1), 1:10 (volume diluent:volume protein solution)(Group-2), 1:100 (Group-3), and 1:1000 (Group-4) compared to two controlgroups; a non-vaccinated challenged group (Group-5) and a non-vaccinatednon-challenged group (Group-6). EMULISIGEN was used as the diluent fordiluting the stock vaccine at a 22.5% concentration prepared inphysiological saline. Mice were vaccinated intraperitoneally andrevaccinated 14 days after the first vaccination. The volumeadministered was 0.25 cc.

Fourteen days after the second vaccination, mice in groups 1–5 wereintraperitoneally challenged with 1.7×10⁸ colony forming units (CFU) ofa virulent Salmonella dublin isolate. The isolate (IRP SCC Serial) wasobtained from The Center of Veterinary Biologics-Laboratory, UnitedStates Department of Agriculture, Ames, Iowa. Mortality was recordeddaily for 2 weeks post-challenge. Table 1 below shows the mortalitybetween the vaccinated and non-vaccinated mice following challenge.

TABLE 1 Mortality of Vaccinated and Non-Vaccinated Mice FollowingChallenge with Salmonella dublin # Percent Groups Mice # Dead mortality(%) Group-1 (non-diluted) 10 0/10 0 Group-2 (1:10) 10 1/10 10 Group-3(1:100) 10 3/10 50 Group-4 (1:1000) 10 5/10 60 Group-5 (non- 10 10/10 100 vaccinated/challenged Group-6 (non-vaccinated/ 10 0/10 0non-challenged

Ten (100%) of the non-vaccinated mice (Group-5) died within 14 daysafter challenge (Table 1). In contrast, none of the mice died given thenon-diluted vaccine of group-1. All dilutions of the test vaccine showeda high degree of protection as compared to the non-vaccinated/challengedmice of group-5. None of the mice died in group-6 showing no horizontaltransmission of the organism between groups.

Example 3 Preparation of an Immunizing Composition Derived fromSalmonella bredeney

Salmonella bredeney was isolated and serotyped from a Minnesota dairyherd having a history of high adult and calf mortality, morbidity andloss of production due to this bacterial strain, and designatedMS010914. SRPs and porins were isolated as described in Example 1. Threehigh molecular weight SRPs, 89 kDa, 84 kDa, and 72 kDa, were observed ona 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-Page) gel. Three additional lower molecular weight iron-regulatedproteins (IRPs) were also isolated at approximately the 37 kDa, 32 kDaand 29 kDa regions. Porins having a molecular weight in the range of38–39 kDa were also purified from the propagated isolates.

Two compositions were prepared from the SRPs having molecular weights of89 kDa, 84 kDa, and 72 kDa, the IRPs having molecular weights of 37 kDa,32 kDa, and 29 kDa, and porins having molecular weights of 38–39 kDa.The target proteins were emulsified in the following vaccineformulations to provide a total dose of about 1,000 μg. In the firstcomposition, referred to as Vac-1, 50 ml of antigen (4.35milligram/milliliter (mg/ml)) was slowly added while stirring to 40 mlof 25% aluminum hydroxide (Rehydagel-HPA, Reheis, N.J.) prepared in 270ml physiological saline. The antigen/aluminum hydroxide suspension wasstirred for 24 hours at 4° C. The antigen/aluminum hydroxide suspensionwas then emulsified into 40 ml of EMULSIGEN, to give a final dose of1,000 μg total protein in a 2 ml injectable volume.

In the second composition, referred to as Vac-2, 217.25 mg of the SRPantigen was mixed into 270 ml of physiological saline. The antigensolution was emulsified into 80 ml of EMULSIGEN to give a final dose of1,000 μg total protein in a 2 ml injectable volume.

Example 4 Pre-Testing of the Immunizing Compositions

To determine any possible side effects (e.g. reduced milk production,adverse tissue reaction, etc.), the vaccines of Example 3 were firstadministered to cattle from various stages of production: 2 lactatingcows, 2 non-lactating adult cows, and 2 calves. Two days prior topre-testing, two lactating cows were selected to determine their dailymilk production. Milk production from each cow was also monitored ateach of the two daily milkings for two consecutive days (48 hours) aftervaccination to determine any loss in production due to vaccination.Monitoring was repeated on a single milking from each cow on day 7. Thetwo lactating cows received 2 mls of Vac-1 subcutaneously in the neckregion. In addition, another 2 non-lactating cows were administered 2.0ml of Vac-2 subcutaneously in the neck region and two calves wereadministered 1.0 ml of Vac-1 subcutaneously in the neck region and theanimals monitored for 7 days for any adverse reaction.

No adverse tissue reactions were observed at any of the injection sitesof the 6 animals given the pre-test vaccines. In addition, there was nomeasurable loss in milk production from the lactating cows at 2 and 7days after vaccination.

Example 5 Herd Immunization

After completion of the study of Example 4, immunizing compositions ofVac-1 and Vac-2 were administered to the entire herd. The herd consistedof 55 lactating cows, 52 non-lactating cows and 18 calves ranging in agefrom 6 months to 12 months. Lactating cattle received 2.0 ml of Vac-1;non-lactating cattle received 2.0 ml of Vac-2; calves less than 12months of age but older then 6 months received 1.0 ml of Vac-1; andcalves greater then 12 months of age received 1.0 ml of Vac-2 (see Table2). All injections were delivered subcutaneously in the neck region.

TABLE 2 Schedule of events. STUDY DAY DESCRIPTION OF EVENTS Pre-testingVaccinated 2 lactating cows, 2 non-lactating cows and 2 calves.Monitored milk production and adverse reactions. First vaccination Day 0Vaccinated all lactating and non-lactating cows (2 ml) except forcalves, under 12 months, gave 1 ml and 2 ml if older then 12 months.Collected blood and fecal samples from lactating cows. Week 3 Collectedblood and fecal samples from lactating cows, examined injection sites.Second vaccination Week 5 Vaccinated all lactating and non-lactatingcows (2 ml) except for the calves, under 12 months, gave 1 ml and 2 mlif older then 12 months. Week 7 Collected blood, fecal samples andexamine injection sites. Week 11 Collected blood, fecal samples andexamine injection sites. Third vaccination Week 19 Vaccinated alllactating and non-lactating cows (2 ml) except for the calves, under 12months, gave 1 ml and 2 ml if older then 12 months. Week 21 Collectedblood, fecal samples and examine injection sites. Week 35 Collectedblood and fecal samples Week 44 Collected blood and fecal samples

Thirty five days after the first vaccination all animals wereadministered a second dose (booster) subcutaneously in the neck. For thebooster dose, all lactating cows received 2.0 ml of Vac-1, non-lactatingcows received 2 ml Vac-2, calves between 6–12 months of age received 1.0ml of Vac-1, and animals 12 months of age or older received 1.0 ml ofVac-2. The schedule of events is shown in Table 2.

Based on the lack of reaction and observed safety of the immunizingcompositions, the herd was vaccinated a third time, 19 weeks after thefirst vaccination (Table 1). The target proteins were emulsified into asingle formulation used in all cows, referred to here as Vac-3. Briefly,300 mg antigen (SRP and porins) was mixed into 250.96 ml ofphysiological saline. The antigen solution was emulsified into 80 ml ofEMULSIGEN to give a final dose of 1,000 μg total protein at a 22.5%EMULSIGEN concentration in a 2 ml injectable volume. All lactating andnon-lactating cows received a 2 ml intramuscular injection while calves6 months of age and older received a 1 ml intramuscular injection.

Example 6 Blood and Fecal Sample Collection and Somatic Cell Counts

Blood samples were collected from twenty lactating cows on the initialday of immunization (day 0) and again at 3, 7, 11, 21, 35 and 44 weeksafter the initial immunization. In addition, fecal samples were takenfrom all lactating cows on the day of immunization (day 0) and again at3, 7, 11, 21, 35 and 44-weeks after immunization (Table 2).

All blood was collected in sterile 13×75 millimeter (mm) vacutainercollection tubes, brand SST No. 369783, (Becton Dickinson, FranklinLakes, N.J.). After clotting, the blood tubes were centrifuged at 800×gfor 30 minutes and frozen at −20° C. until analysis.

Individual fecal samples were taken aseptically by rectal extractionusing sterile shoulder length gloves and placed in sterile whirl packbags. Ten grams of feces from each sample was placed into 90 ml ofTetrathionate broth (Difco) and incubated at 37° C. for 24 hours. Eachsample was plated onto Bismuth sulfite, Brilliant green and XLD agar(Difco) as a differential selective media to identify the presence ofSalmonella. All suspect isolates were confirmed to be Salmonella usingSalmonella O antiserum (poly A-I and Vi) with a slide agglutinationtest. Briefly, a colony is removed from a plate and mixed in a drop ofpoly O antiserum. This is mixed for about 30 seconds if it agglutinatesit's a confirmed suspect. Confirmed Salmonella isolates were sent to theMinnesota Poultry Testing Laboratory (MPTL), Willmar, Minn., forserotyping.

Somatic cell counts were per milliliter of milk were conducted by theDairy Herd Improvement Association (DHIA, Buffalo, Minn.) using standardmethods. The somatic cells counted were the white blood cells present inthe milk.

Example 7 Enzyme-Linked Immunosorbent Assay (ELISA)

An Enzyme-Linked Immunosorbent Assay (ELISA) monitored the serologicalresponse to the vaccine. The highly conserved SRPs from Salmonellabredeney having molecular weights of 89 kDa, 84 kDa, and 72 kDa werepurified from polyacrylamide gels. Briefly, the corresponding SRP bands(89 kDa, 84 kDa, and 72 kDa) were cut from unstained gels using astained indicator lane for determining band location which was cut awayfrom the original gel and stained. Elution of the protein from themacerated gel was carried out according to the manufacturesrecommendation using a model 422 electro-eluter (Bio-Rad, Laboratories,Hercules, Calif.). These proteins were then used as the capture moleculein an indirect ELISA test.

Polyclonal antiserum was raised against the vaccine composition ofexample 4. Briefly, the vaccine composition consisting of SRPs andporins of Salmonella bredeney was inoculated subcutaneously into 2 adultHolstein heifers (2 ml dose at 1000 ug total protein). Each Heiferreceived a total of three vaccinations 21 days apart. Fourteen daysafter the third vaccination 20 ml of blood was collected from the tailvein of vaccinated cows. In addition negative control serum (20 ml) wasobtained from two non-vaccinated cows. The hyperimmune and negativeserum was obtained by centrifugation (800×g) of the clotted blood. Thehyperimmune and control sera was absorbed with killed whole cellbacteria of Salmonella bredeney grown in iron-replete media (BHIcontaining 200 um ferric chloride) for 1 hour at 4° C.

Twenty milliliters of the positive and negative control sera wasprecipitated for 6 hours using ammonium sulfate (60% saturation),dissolved in 0.02 M phosphate buffer pH 7.0 at 4° C. The precipitate wascollected by centrifugation at 8000×g for twenty minutes. The pellet wasresuspended in 20 ml of 50 mM phosphate buffer pH 7.2 and dialyzed usinga 100,000 MWCO dialysis tubing (Pierce, Rockford, Ill.) against 0.02 MpH 7.2. The dialyzed material was concentrated 10 times using a Diafloultrafiltration apparatus model 8200 with a 50,000 MWCO membrane(Amicon). The positive and negative control dialysate was alquoted into100 ul samples and frozen at −90° C.

The optimum working concentrations of SRP and conjugate was determinedby several checkerboard titrations using the positive and negativecontrol dialysates. A prediction curve was then established to calculateSRP ELISA titers at a 1:500 dilution. All subsequent tests wereperformed at a single serum dilution (1:500) and SRP titers werecalculated from the average of duplicate test absorbance values.

The ELISA was performed by adding 100 ul of diluted SRP of Salmonella in0.05 M carbonate buffer (pH 9.6) to each well of a 96-well flat bottom,easy wash microtiter plate (Corning, Corning N.Y.). After overnightincubation at 4° C., excess SRP was removed and the plate was washed.All subsequent washing steps were done three times in phosphate bufferedsaline (pH 7.4) with 0.05% Tween-20. The plates were blocked for onehour at 37° C. with 4% fish gelatin (Sigma) in PBS and then washed.

Duplicate serum samples from Example 4 were tested in parallel atsingle-point dilutions using 100 ul/well and incubated for 45 minutes at37° C. The first two rows of each plate contained the negative andpositive control samples while the rest of the plate was used for thetest samples. The plate was incubated for 45 minutes at 37° C. whilestirring at 200 rpm. After washing, 100 ul alkaline phosphataseconjugate (Monoclonal anti-bovine IgG clone BG-18, Sigma) at a 1:15,000dilution was added to each well. After incubation for 45 minutes at 37°C., the plates were washed and 100 ul p-NitroPhenyl Phosphate (pNPP)substrate (Sigma) was added to each well. The substrate was allowed toreact for 2 hours at 37° C. while stirring at 100 rpm. The reaction wasterminated by the addition of 25 ul of 3N NaOH. The absorbence was readat 405 nm.

Results of Examples 3–7

FIG. 1 shows the cumulative history of the shedding prevalence ofSalmonella compared to the serological response to vaccination inlactating cows. As described in Example 5, the herd was vaccinated onthe day of the initial immunization (Day 0) and again at 5 and 19 weeksafter the first vaccination. Fecal and blood samples were taken from alllactating cows at 0, 3, 7, 11, 21, 35, and 44 weeks. Briefly, theimmunizing compositions consisting of Vac-1 and Vac-2 were given to allcows (N=125) in the herd on the day of the initial immunization, day 0(Table 1). Only the lactating cows were monitored through theexperimental trial. All lactating cows were subcutaneously given 2 ml ofVac-1. The shedding prevalence of Salmonella in the fecal samples takenfrom the lactating cows (N=55) on day 0 revealed an isolation rate of85.4% (FIG. 1). All of the Salmonella isolates were serotyped and foundto be S. bredeney.

Within this same time period the somatic cell count as determined byDHIA was 1,492,000 cells per milliliter of milk (Table 3), the highestit had ever been in the history of the farm.

TABLE 3 The Somatic Cell Count (SCC)¹ of Individual Cows Before andAfter Vaccination SCC before/after vaccination² SCC before/aftervaccination Cow ID SCC × 1000 Cow ID SCC × 1000 Cow ID SCC × 1000 Cow IDSCC × 1000 1 1980/3930 14 570/680 27 970/160 40 40/50 2 9990/3870 15 63/520 28 1150/130  41 140/50  3 1230/3370 16 460/460 29 140/120 42210/40  4 1240/2090 17 9990/450  30  50/120 43 70/40 5 4390/1980 183890/360  31 660/120 44 80/30 6 5510/1660 19 160/350 32 450/120 45 90/307 2090/1550 20 570/290 33 380/110 46 110/30  8  870/1170 21 7070/250  34220/100 47 230/30  9 3020/960  22 210/240 35 350/100 48 3200/20  10 2620/950  23 230/220 36 70/90 49 20/20 11  1040/780  24  50/210 37890/60  50 50/20 12  1330/720  25 190/190 38 700/60  51 40/10 13 120/720 26 540/180 39 100/50  Average SCC 1492/585 ¹Number of somaticcells per milliliter of milk. ²Samples taken before vaccination weretaken in July (year 2, immediately before vaccination), and samplestaken after vaccination (year 2) were taken in August (P = 0.0068).

Three weeks after the first vaccination, fecal samples taken from alllactating cows (N=54) revealed no significant change in the sheddingprevalence of Salmonella, which remained at 87%, (FIG. 1). Nevertheless,the somatic cell count dropped to 585,000 cells per milliliter. This isgraphically and numerically depicted in FIG. 2 and table 3 which showsthe DHIA somatic cell count on individual cows before and after thefirst vaccination. There was a 61.0% drop in somatic cell count having adegree of significance of P=0.0068. This highly significant affect wasobserved without improvements in management and/or environmentalchanges. One year after the first vaccination the cumulative 12 monthaverage in somatic cell count was 417,000 cells per milliliter of milk.In contrast, the 12 month average before vaccination was 660,000 somaticcells per milliliter of milk. This was a 37% decrease in the somaticcells after vaccination. It is interesting to speculate that because ofthe conserved nature of these proteins it induced a degree ofcross-protection against other gram negative or gram positive bacteriaresponsible for contagious and/or environmental mastitis.

The injection sites of all calves and lactating cows were examined 14days after the first vaccination. None of the cows examined showed anyadverse tissue reaction at the site of injection by physicalexamination. In addition, there was no measurable loss in milkproduction due to vaccination.

Five weeks after the first vaccination the herd was given a booster(Table 2). Fourteen-days after the second vaccination (Week 7) there wasa 21.2% drop in the shedding prevalence of Salmonella with the totalnumber of isolations being 35 out of 54 samples taken or, 64.8% of theherd positive for Salmonella in contrast to a previous prevalence of 86%(FIG. 1). The isolation rate continued to decline and by the eleventhweek the shedding prevalence was 47.1% or 24 positive isolates out of 51cows sampled. This was a 52.9% reduction in the number of positiveSalmonella isolations. Physical examination of the injection sitesshowed no adverse tissue reaction in any of the calves and/or lactatingcows examined. However, the second vaccination resulted in approximatelya 2% drop in milk production that began 24 hours after vaccination butlasted less than two days. At this point the data showed the vaccinecompositions to be highly tissue compatible with minimal loss in milkproduction. In addition, the data indicated a direct correlation betweenthe declining shedding prevalence of Salmonella to the increasing SRPantibody response.

To stimulate a higher SRP antibody response the herd was vaccinated athird time, fourteen weeks after the second vaccination (Week 19, Table2). The protein concentration of the vaccine remained the same (1000μg/2 cc dose) but the adjuvant (EMULSIGEN) was increased to 22.5%vol/vol. Blood and fecal samples were taken 14-days after vaccination(Week 21, Table 2). The shedding prevalence of Salmonella declined to45%, i.e., only 28 cows out of 61 sampled were positive for Salmonella(FIG. 1). All of these samples were serotyped and found to be Salmonellabredeney. At this same time period the injection sites of each lactatingcow was examined, and less than 5% developed a granuloma that measuredapproximately 1 centimeter×1 centimeter. These granulomas resolvedwithin 21 days after injection.

The cumulative pounds of milk produced before and after the thirdvaccination is shown in FIG. 3. After the third vaccination the drop inmilk production peaked at 6.9%. This loss in production appearedtransient within the herd, lasting less then four days, at which pointthe herd regained normal production. In fact, after the thirdvaccination the average milk production for the remainder of the monthincreased by 1.2% as compared to production before vaccination (FIG. 3).This increase in milk production was consistent and started at thebeginning of the first vaccination. For example, the DHIA rolling herdaverage for 45 cows for the month of December (year 1, beforevaccination) was 16,787 pounds of milk (FIG. 4 and Table 4). The generalhealth and overall performance of the herd increased after eachvaccination. Fourteen days after the third vaccination (December) therolling herd average for 53 cows was 18,047 pounds of milk produced(FIG. 4 and Table 3). This was a 7.0% increase in milk per cow or andaverage of 1,260 pounds per year. In addition, the annual pounds of milkproduced 1 year after vaccination was 965,472 pounds compared to 740,855pounds produced before vaccination. This was a 6% increase in the totalpounds of milk produced.

TABLE 4 The Annual Herd Summary From the Onset of the First Salmonellaisolation DHI Rolling Herd Average-Entire Herd Year Sampled (year 1)Year Sampled (year 2) Year Sampled (year 3) Date¹ DIM² Milked³ Lbs.⁴Date DIM Milked Lbs. Date DIM Milked Lbs. Jan 183 49 15563 Jan 201 5016535 Jan 241 55 18703 Feb 170 58 15795 Feb 171 57 16258 Feb 260 5418776 Mar 173 56 15725 Mar 155 62 16310 Mar 251 54 18516 Apr 192 5315643 Apr 163 60 16409 Apr 227 55 18261 May 217 51 15584 May 157 6316421 May 220 52 18068 Jun 229 49 15467 Jun 165 64 16574 Jul 244 5115503 Jul 161 56 16849 Aug 280 51 15841 Aug 182 56 17080 N/A N/A N/A N/ASept 207 56 17329 Oct 300 48 16315 Oct 231 54 17570 Nov 291 48 16606 N/AN/A N/A N/A Dec 264 45 16787 Dec 229 53 18047 ¹Date: year 1, year beforevaccination; year 2, year during which cows were vaccinated; year 3,year after cows were vaccinated. ²DIM, days in milk. ³Milked, number ofcows milked during the time period. ⁴Lbs., total pounds of milk producedduring the time period.

FIG. 5 shows the average monthly cost in antibiotic usage calculated 12months after the first vaccination compared to 12 months beforevaccination. The average monthly cost in antibiotic usage beforevaccination or during the course of Salmonellosis was $284.65 comparedto $144.05 after vaccination. This was a 51% reduction in the cost ofantibiotics.

At the onset of the first isolation of Salmonella bredeney (January,year 1) and clinical diagnosis from this herd, approximately 21 adultcows and 36 calves died of clinical Salmonellosis. This mortalityoccurred despite vaccinating the herd three separate times over a oneyear period, using a commercial whole cell bacterin of Salmonella dublinand Salmonella typhimurium. The herd was up to date in all, routineviral and bacterial vaccines. After the first vaccination with thecomposition described in Example 3, mortality and morbidity virtuallyceased. Six months after the first vaccination only three calves diedwithin this time period. None of the calves that died within this timeperiod were diagnosed with Salmonella. There has been no mortality inany of the non-lactating (dry), lactating cattle and/or calves in thisherd, since the first Salmonella vaccination. From this data it wouldappear the vaccine induced a high degree of humoral immunity againstfield challenge as well as providing passive immunity to newborn calves.It was also apparent in this field study that as the serologicalresponse to vaccination increased, the shedding prevalence of Salmonelladecreased. It is interesting to note that the antibody titer continuedto rise 25 weeks after the third vaccination. This continued rise intiter could be due to clinical field challenge by Salmonella or othergram negative bacteria expressing these highly conserved proteins duringsubclinical infections.

Vaccination improved the overall health and performance status of theherd as observed by the decrease in mortality, decreased somatic cellcounts and the increase in milk production. Calf health also improved,as calves were more active at birth, consumed colostrum aggressively anddid not develop any significant diarrhea symptoms. In addition, therewas an observed decrease in clinical metritis in the fresh cows thatwere brought back into production after calving. These cows werevaccinated at dry off and boosted prior to calving. Vaccination appearedto alleviate the incidence of clinical metritis during the post-calvingperiod. This was initially observed while taking fecal samples for theisolation of Salmonella in that rectal palpation of the uterus could bedone at the same time. The incidence of metritis dramatically decreasedafter vaccination as compared to previous years.

The vaccine composition proved to be highly tissue compatible. None ofthe vaccinated cows showed any adverse tissue reaction at the site ofinjection or any physical signs of stress such as, depression, lethargy,loss of milk production, etc. The compatibility of the vaccinecomposition is likely due to its purity and lack of contaminatinglipopolysaccharides (LPS). LPS has been shown to be responsible for muchof the tissue reactions in conventional vaccines, such as whole cellbacterins. The concentration of LPS in the stock antigen of Example 3was found to be negative as examined by the Limulus Amebocyte LysateAssay (SIGMA, Chemical Company, St Louis Mo.).

Example 8 The Effect of Vaccinating Fresh Cows and Cows in FirstLactation

A subunit vaccine consisting SRPs and porins derived from Salmonelladublin (strain designation MS010207) and Salmonella typhimurium (straindesignation MS010427) were administered to two groups of lactating cowsin a controlled field study within a large expansion dairy. The dairyconsisted of 500 cows separated into five large freestall corrals (100cows/corral) based on days in milk or period of lactation. Two groups ofcows were chosen for the study; fresh cows (30–90 days post-partum) andhigh-producing heifers (cows in first lactation). Cows received twosubcutaneous vaccinations 28 days apart. The experimental trial examinedthe safety of the immunizing composition based on the tissue reactivityof the injected material at the site of injection, the effectvaccination had on milk production, the prevalence of Salmonella andsomatic cell counts between vaccinated and non-vaccinated cows. Data wascollected on performance and physiological status from individual cowsusing an integrated electronic cow identification system.

Preparation of an Immunizing Composition Derived from Salmonella dublinand Salmonella typhimurium

The immunizing composition was prepared as described in Example 3 withthe following modifications. Three high molecular weight SRPs atapproximately the 89 kDa, 84 kDa and 72 kDa and porins in the range of38–39 kDa were harvested from each of the two isolates. The lowermolecular weight IRPs (37 kDa, 32 kDa and 29 kDa) that S bredeneyexpressed under iron restriction of Example 3 were poorly expressed inS. dublin and/or S. typhimurium and were not present in the final stockantigen as examined on a 10% SDS-Page gel. Nevertheless, the upperbanding profile (89 kDa, 84 kDa and 72 kDa) of these two isolates wereidentical to S. bredeney of Example 3. The immunizing compositionconsisted of equal concentration of SRPs from S. dublin and Styphimurium so as to provide a total dose of 1000 μg, 500 μg from eachisolate. The antigen solution was emulsified into EMUSIGEN (22.5%vol/vol) as previously described in Example 3.

Pre-Vaccination

Thirty days before the first vaccination the herd-exposure status toSalmonella was determined. Fecal samples were collected from eachindividual cow as described in Example 6. The total number of samplescollected was 144 (60 Fresh cows and 84 Heifers). Salmonella wasrecovered from 50% of the Fresh cows and 27% from the cows in firstlactation. Three serotypes were found; S. anatum, S. uganda and S.meleagridis; S. dublin and S. typhimurium were not detected. The SRP andporin profiles of these isolates were found to be identical to thebanding profiles of S. dublin, S. typhimurium and S. bredeney. Becauseof the wide spread incidence of S. dublin and S. typhimurium in thebovine species and the conserved nature of these proteins it was decidedto use these antigens in the vaccine composition to give furtherclarification of the cross-protective nature of these proteins.

Immunization of Fresh Cows and Cows in First Lactation

Fifty percent of the cows in first lactation (42 out of 84) and 50% ofthe fresh cows (30 out of 60) were vaccinated. The remaining cows ineach group remained as non-vaccinated controls. Briefly, cows from eachgroup were randomly placed in a large holding stanchion. Every other cowwas given a 2 ml intramuscular injection of the vaccine. In addition,fecal samples were taken from all cows in each group by rectalextraction at the time of the first vaccination. All suspect isolationswere serotyped as described in Example 6. The somatic cell count andmilk production for each cow was acquired prior to the first vaccinationto establish a historical performance trend. The production of milk fromindividual cows was monitored daily as well as general health andadverse reaction to vaccination. The somatic cell counts were monitoredmonthly by the DHIA. The vaccinated cows were given a second vaccination(Booster) four weeks after the first vaccination. The vaccinated andnon-vaccinated test cows within the herd were identified by ear tags andmilk production was monitored by an electronic cow identification systemusing a transponder, hung on a strap around the cows neck. The overallperformance of the vaccinated and non-vaccinated cows was monitoredthroughout the experimental study.

Results

The injection sites of vaccinated cows were examined 14 days after thefirst and second vaccination. None of the vaccinated cows showed anyadverse tissue reaction to the vaccine at the site of injection. Therewas no visible swelling or defined nodule in any of the cows examined.In addition, daily observations of these cows showed no visible changesin behavior and/or activity.

Fecal samples taken from both groups the day of the first vaccinationrevealed a significant decline in the shedding prevalence of Salmonellaas compared to samples taken 30 days before vaccination. The isolationrate in the fresh cow group declined to 27% while the cows in firstlactation had dropped to 8%. In fact, the isolation rate of Salmonellaat the second vaccination showed no difference between groups. Only fiveisolates of Salmonella were cultured between groups, three from thefresh cow group and 2 from the first lactation cow group. There was nodifference in the shedding prevalence of Salmonella between thevaccinated and non-vaccinated cows from either group.

The yield of milk per cow was monitored daily in both groups. FIG. 6shows the weekly average milk production between the vaccinated andnon-vaccinated cows in first lactation. There was no statisticaldifference in the yield of milk from the first vaccination (week 1) tothe second vaccination (week 2) when compared to the non-vaccinated cows(P=0.435) and from the second vaccination (week 5) through the 16^(th)week of production (P=0.07) as graphically depicted in FIG. 6. However,in the fresh cow group, the production of milk statistically increasedin the vaccinated cows after each vaccination as compared to thenon-vaccinated group (FIG. 7). The degree of significance from the firstvaccination to the second vaccination was P=0.006 and dramaticallyincreased from the second vaccination to the 16^(th) week of production(P=0.000000067). Sixteen weeks after the first vaccination the averagepounds of milk produced per cow in the vaccinated group was 60.3 poundscompared to 56.4 pounds in the non-vaccinated controls. This was a 6.5%increase in milk production over the control group or 3.9 pounds/cowadvantage.

The somatic cells counts were also positively effected throughvaccination in both the fresh cows and cows in first lactation. FIG. 8shows the monthly average (DHIA) somatic cell counts between thevaccinated and non-vaccinated cows in first lactation, beginning fromthe first vaccination through 16 weeks of production. The data showsthat the vaccinated group had a 30.0% difference in the average somaticcell count with a degree of significance of P=0.036 as compared to thenon-vaccinated control group. This reduction in somatic cell count wasmore dramatically pronounced in the vaccinated fresh cows as illustratedin FIG. 9. The data shows that the level of somatic cells decreased by58.8% (P=0.02) in the vaccinated cows as compared to the non-vaccinatedgroup.

The difference in performance between the fresh cows and in firstlactation could be due to the difference in the health status of thecow. Typically fresh cows are under a higher degree of stress due totheir physiological status then other cows in production, predisposingthem to a greater disease challenge. Stress can often exasperate thelikelihood of a diseased condition that may effect the overall healthand performance of the animal. It is interesting to note there was nostatistical difference in milk production between the vaccinated andnon-vaccinated cows in first lactation, in contrast to the vaccinatedfresh cows. It would appear that the vaccine had a positive effect onthe health status of the vaccinated fresh cows, as seen by the enhancedmilk production. This enhanced performance did not appear to be relatedto a clinical disease caused by Salmonella since the isolation ratenaturally declined and there was no difference in the prevalence betweenvaccinated and non-vaccinated cows. In addition, the vaccine compositioncontained the immunogens derived from Salmonella dublin and Salmonellatyphimurium and not from the isolates found within the herd. Because ofthe conserved nature of these proteins among gram negative and grampositive bacteria it is highly likely that the vaccine induced a degreeof cross-protection against other bacteria expressing these proteins,allowing the animal to perform better.

Example 9 Immunization of Feed Lot Steers for the Control of Salmonella,Trial-1

A commercial feed lot having a history of Salmonellosis was used in acontrolled field study to evaluate the efficacy of an immunizingcomposition consisting of SRPs and porins derived from Salmonelladublin. The experimental trial examined the safety of the immunizingcomposition based on the tissue reactivity of the injected material atthe site of injection, the serological response to vaccination, and theshedding prevalence of Salmonella.

The feed lot consisted of 500 Holstein steers separated into separategrow out facilities based on the age and weight of the steers. Theexperimental trial was initiated in starter calves (N=150) with anaverage weight of approximately 150 pounds. The steers were randomlydistributed into 10 separate pens (1–10) so that each pen contained 15steers. Ear tags individually identified steers in each pen. Theexposure status to Salmonella was determined prior to the firstvaccination. Individual fecal samples were taken from all steers toestablish a shedding prevalence of Salmonella. Samples were processed aspreviously described in Example 6. Salmonella was recovered from 56% ofthe 150 samples taken. Three different serotypes were identified; S.dublin, S. uganda and S. muenster. Salmonella dublin was the predominantserotype, and was found within the herd at 67%.

The Salmonella positive steers were identified from each pen anddistributed among four pens (P3, P4, P5, and P6) so that each pencontained the same number of positive and negative steers. Thus, eachpen contained 8 Salmonella positive steers and 7 Salmonella negativesteers so that 53.3% of each pen was Salmonella positive.

The immunizing compositions was prepared from the SRPs of S. dublinhaving molecular weights of 89 kDa, 84 kDa, 72 kDa and porins havingmolecular weights of 38–39 kDa. The target proteins were emulsified intoEMULSIGEN (22.5% vol/vol) to provide a total protein dose of a 1000 μgin a 2 ml injectable volume as previously described.

Steers in pens 3 and 5 received two intramuscular vaccinations 28 daysapart. Steers in pens 4 and 6 remained as non-vaccinated controls. Bloodwas taken from 12 steers per pen at day 0 (First vaccination) and againat 2, 4, and 6 weeks post to monitor the serological response tovaccination. Individual fecal samples were collected from each steer asdescribed in Example 6 on the day of the first vaccination and again at2 and 6 weeks.

The injection sites of each vaccinated steer were examined 14 days afterthe first and second vaccination. None of the vaccinated steers showedany adverse tissue reaction at the site of injection. In addition, dailyobservations of these steers showed no visible changes in behaviorand/or activity as compared to the non-vaccinated groups. FIG. 10 showsthe serological response of vaccinated steers compared to non-vaccinatedcontrols as evaluated by ELISA of Example 7. The vaccine inducedelevated antibody titers to SRPs after each vaccination. There was arise in titer after the first vaccination that declined two weeks afterbut continued to rise after the second vaccination, clearlydemonstrating that a secondary response was induced.

Table 5 shows the shedding prevalence of Salmonella between vaccinatedand non-vaccinated pens. Fecal samples taken from individual steers onthe day of the first vaccination (week 0) revealed a significant declinein the shedding prevalence of Salmonella in all test groups as comparedto samples taken before vaccination (Table 5). The decline in theshedding prevalence continued through the duration of the samplingperiod in all test groups. However, the shedding prevalence 14 daysafter the last vaccination indicated a difference between the vaccinatedgroup as compared to the non-vaccinated controls. There was a higherpercentage of Salmonella positive steers (29%) in Pen 6 as compared tothe vaccinated pens showing only 6.7%.

TABLE 5 The Shedding Prevalence of Salmonella Between Vaccinated andNon-vaccinated Pens Pen 3- Pen 4 Pen 5 Pen 6 Sampling time VaccinatedControl Vaccinated Control Pre-vaccination 53.3%   53.3%   53.3%  53.3%   0 33% 40% 47% 43% 2 weeks 27% 13% 13% 36% 6 weeks 6.7%  13%6.7%  29%

Example 10 Immunization of Feed Lot Steers for the Control of SalmonellaTrial-2

The commercial feed lot of Example 9 was used in a controlled fieldstudy to provide further data on the efficacy of an immunizingcomposition consisting of SRPs and porins derived from Salmonella dublinand Salmonella typhimurium. The experimental trial examined the safetyof the immunizing composition based on the tissue reactivity of theinjected material at the site of injection, the serological response tovaccination, and the shedding prevalence of Salmonella.

At the end of the experiment of Example 9 the facility was cleaned,sanitized and disinfected and allowed to sit empty for 2 weeks prior tothe arrival of a new group of steers. Environmental samples (N=2) weretaken from each pen to ascertain the incidence of Salmonella. Sampleswere cultured as previously described in Example 6. All environmentalsamples were found negative for Salmonella. One hundred fifty (N=150) 4month old Holstein steers with and average weight of 300 pounds weretransported by truck from Idaho. Upon arrival, steers were unloaded, eartagged for identification and randomly distributed among 10 separatepens (1–10) so that each pen contained 15 steers. One week after arrivalthe exposure status to Salmonella was determined prior to the firstvaccination. Individual fecal samples were taken from all steers toestablish a shedding prevalence of Salmonella. Samples were processed aspreviously described in Example 6. All of the 150 samples taken werefound negative for Salmonella. Based on this information a vaccinecomposition was prepared from two Salmonella isolates (S. dublin (straindesignation MS010207) and S. typhimurium (strain designation MS010427)).

The immunizing compositions was prepared from the SRPs of S. dublin andS. typhimurium having molecular weights within a range of 89 kDa, 84kDa, 72 kDa and porins having molecular weights within a range of 38–39kDa. The proteins (500 μg from each isolate) were absorbed onto aluminumhydroxide (25% vol/vol) to provide a total protein dose of a 1000 μg ina 2 ml injectable volume.

As before, steers in pens 3 and 5 received two intramuscularvaccinations 28 days apart. Steers in pens 4 and 6 remained asnon-vaccinated controls. Blood was taken from 12 steers per pen at day 0(First vaccination) and again at 2, 4, and 6 weeks post to monitor theserological response to vaccination. Individual fecal samples werecollected from each steer as described in Example 6 on the day of thefirst vaccination and again at 2 and 6 weeks.

The injection sites of each vaccinated steer, as before, were examined14 days after the first and second vaccination. None of the vaccinatedsteers showed any adverse tissue reaction at the site of injection usingaluminum hydroxide as the adjuvant. In addition, daily observations ofthese steers showed no visible changes in behavior and/or activity ascompared to the non-vaccinated groups. The serological response to thevaccine was determined as described herein and compared to thenon-vaccinated controls. The vaccine induced elevated antibody titers toSRPs after each vaccination that was comparable to the composition ofExample 9. There was a rise in titer after the first vaccination thatdeclined for two weeks after but continued to rise after the secondvaccination.

Fecal samples were taken from all steers at the time of firstvaccination and again at 2 and 6 weeks after vaccination. Salmonella wasnot isolated from any of the samples taken during the sampling period.In addition, environmental samples (N=2) taken from each pen at the 6week period were negative for Salmonella.

Nine weeks after the first vaccination steers in both the control andvaccinated pens were individually weighed. The average weight of steersin the control pens were 730.5 lbs (Pen-4) and 745.6 lbs (Pen-6) (Table6) with an average weight of both pens at 738.0 lbs. In contrast, theaverage weight of vaccinated steers were 767.7 pounds (Pen-3) and 761.8lbs (Pen-5) (Table 6) with a combined average weight of 764.8 lbs. Therewas a 26.7 pound advantage in the vaccinated steers as compared to thesteers in the non-vaccinated groups with a degree of significance ofP=0.018. This enhanced weight performance did not appear to be relatedto a clinical disease caused by Salmonella since the organism was notdetected in any of the steers examined. It is believed that theconserved nature of these proteins in the vaccine composition induced adegree of cross-protection against other bacteria expressing theseproteins, thus lessening subclinical diseases, allowing the animal toperform better as seen in the difference in weight between the twogroups.

TABLE 6 The Comparison of Individual Weights Between Vaccinated andNon-Vaccinated steers 9 weeks after the first vaccination Pen-3 Pen-4Pen-5 Pen-6 Vaccinated Control Vaccinated Control Weight in Weight inWeight in Weight in pounds Pounds Pounds Pounds 742 682 816 724 889 723717 812 750 595 716 756 712 705 811 735 844 737 801 779 794 726 740 717769 780 796 744 755 752 758 670 698 811 785 749 772 706 785 775 746 778743 729 744 725 764 819 697 741 719 712 809 744 775 688 795 752 701 775Mean = 767 Mean = 730.5 Mean = 761.8 Mean = 745.6 SD¹ = 52.7 SD = 49.7SD = 37.6 SD = 41.9 CV = 6.9 CV = 6.8 CV = 4.9 CV = 5.7 ¹SD, standardvariation. ²CV, coefficient of variation.

Example 11 Purification of Siderophore Receptor Proteins ofStaphylococcus aureus of Human and Avian Origin

Two field isolates of Staphylococcus aureus and three additionalisolates obtained from the American Type Culture Collection ATCC(isolates 8432, 11371, and 19636) were evaluated for the expression ofsiderophore receptor proteins. Field isolates originating from turkeyswere isolated from the hock joints of diseased birds. ATCC isolate 8432was also of avian origin, while isolates 11371 and 19636 were of humanorigin. All bacteria were grown in Brain heart infusion broth (BHI,Difco) as iron-deplete and/or iron-replete media. The iron-deplete mediawas iron-restricted chemically using 2′2′-dipyridyl at 175 mM, whereasthe iron-replete media contained 200 μM ferric chloride. The bacteriawere grown in 10 ml of BHI for 8 hours at 37° C. while stirring at 400rpm. At 8 hours of incubation, 1.0 ml of culture was removed and washedin 10 volumes sterile physiological saline by centrifugation (10,000×g)for 10 minutes. The pellet was resuspended in 100 microliters (μl) ofsaline containing 1 mg lysostaphin (Sigma, St. Louis, Mo.) was added,and the suspension was then incubated at 37° C. for 2 hours. Thebacterial suspension was centrifuged at 12,000×g for 1 minute. Thesupernatant was collected and centrifuged again at 20,000×g for 40minutes. The bacterial pellet was resuspended in 100 μl tris-bufferedsaline (TBS) at pH 7.4. The resuspended bacterial pellets from thedifferent isolates were resolved by 12% SDS-PAGE and transferred ontonitrocellulose membranes and tested for cross-reactivity with sera toSRPs of gram negative bacteria. Absorbed rabbit polyclonal hyper-immunesera prepared against purified SRPs from E. coli and/or S. typhimuriumwere used as probes in the immunoblot of the S. aureus SRPs.

The SDS-PAGE patterns of the outer membrane protein extracts of theStaphylococcus aureus isolates showed different patterns of SRPexpression between the field isolates and the ATCC isolates. The fieldisolates of turkey origin grown under conditions of iron restrictionshowed four proteins with molecular weights between 66–90 kDa(specifically, 90 kDa, 84 kDa, 72 kDa and 66 kDa) and also at about 36kDa, 32 kDa and 22 kDa regions. The ATCC isolates showed only a singleSRP at the 40–55 kDa range (42 kDa) and at the 36 kDa range. None of theATCC isolates showed an SRP at 66–90 kDa region, including isolate 8432of avian origin.

Western blot analysis of the isolated SRPs of the S. aureus fieldisolates was conducted by probing with sera raised to the SRPs ofSalmonella (89 kDa, 84 kDa and 72 kDa) and/or E. coli (89 kDa, 84 kDa,78 kDa, and 72 kDa). The sera reacted strongly with the proteins in the66–90 kDa range but not with the lower molecular weight proteins (i.e.,36 kDa, 32 kDa and 22 kDa). The Salmonella sera also reacted with aprotein in the 31 kDa range that appeared to be similar to the 31 kDaprotein of the transmembrane proteins of gram negative bacteria.

This data indicates that S. aureus expressed SRPs that are within asimilar molecular weight range as gram negative bacteria, and thatantibodies raised against SRPs from gram negative bacteria cross-reactbetween at least two different families of bacteria. This composition isused to vaccinate animals as described herein, and the ability of thecomposition to protect animals from homologous and heterologouschallenge is determined, as well as the ability of the composition toenhance performance characteristics of the animal.

The complete disclosure of all patents, patent applications, andpublications, and electronically available material (including, forinstance, nucleotide sequence submissions in, e.g., GenBank and RefSeq,and amino acid sequence submissions in, e.g., SwissProt, PIR, PRF, PDB,and translations from annotated coding regions in GenBank and RefSeq)cited herein are incorporated by reference. The foregoing detaileddescription and examples have been given for clarity of understandingonly. No unnecessary limitations are to be understood therefrom. Theinvention is not limited to the exact details shown and described, forvariations obvious to one skilled in the art will be included within theinvention defined by the claims.

All headings are for the convenience of the reader and should not beused to limit the meaning of the text that follows the heading, unlessso specified.

1. A method for treating an animal having low milk production, themethod comprising administering to a milk producing animal having or atrisk of having a low milk production an effective amount of acomposition comprising: at least two siderophore receptor polypeptidesisolated from a gram negative microbe; at least two porins isolated fromthe gram negative microbe; lipopolysaccharide at a concentration of nogreater than about 10.0 EU/m1; and a pharmaceutically acceptablecarrier.
 2. The method of claim 1 wherein the gram negative microbe isan enteropathogen.
 3. The method of claim 1 wherein the gram negativemicrobe is a member of the family Enterobacteriaceae.
 4. The method ofclaim 1 wherein the gram negative microbe is a member of the tribeEscherichieae or Salmonelleae.
 5. The method of claim 1 wherein the gramnegative microbe is Salmonella spp. or Escherichia coli.
 6. The methodof claim 1 wherein the at least two siderophore receptor polypeptideshave molecular weights of between about 60 kDa and about 100 kDa asdetermined by separation by sodium dodecyl-polyacrylamide gelelectrophoresis.
 7. The method of claim 1 wherein the at least twoporins have molecular weights of between about 30 kDa and about 43 kDaas determined by separation by sodium dodecyl-polyacrylamide gelelectrophoresis.
 8. The method of claim 1 wherein the animal is abovine.
 9. A method for treating an animal having low milk production,the method comprising administering to a milk producing animal having orat risk of having a low milk production an effective amount of a firstcomposition comprising: at least two siderophore receptor polypeptidesisolated from a gram negative microbe; at least two porins isolated fromthe gram negative microbe; lipopolysaccharide; and a pharmaceuticallyacceptable carrier, wherein the concentration of lipopolysaccharidepresent in the first composition is no greater than the concentration oflipopolysaccharide in a second composition comprising: the at least twosiderophore receptor polypeptides isolated from the gram negativemicrobe; the at least two porins isolated from the gram negativemicrobe; lipopolysaccharide; wherein the second composition is producedby a process comprising: providing the gram negative microbe; disruptingthe gram negative microbe in a buffer; solubilizing the disrupted gramnegative microbe for greater than about 24 hours in a solutioncomprising sarcosine to result in solubilized and insoluble cellularmaterial, wherein a ratio of the sarcosine to gram weight of disruptedgram negative microbe is between about 0.8 gram sarcosine per about 4.5grams of disrupted gram negative microbe and about 1.2 grams sarcosineper about 4.5 grams of disrupted gram negative microbe; and isolatingmolecules of the gram negative microbe, wherein the isolated moleculescomprise the at least two siderophore receptor polypeptides, the atleast two porins , and lipopolysaccharide.
 10. The method of claim 9wherein the isolated molecules further comprise lipopolysaccharide at aconcentration of no greater than about 10.0 EU/ml.
 11. The method ofclaim 9 wherein the gram negative microbe is an enteropathogen.
 12. Themethod of claim 9 wherein the gram negative microbe is a member of thefamily Enterobacteriaceae.
 13. The method of claim 9 wherein the gramnegative microbe is a member of the tribe Escherichieae or Salmonelleae.14. The method of claim 9 wherein the gram negative microbe isSalmonella spp. or Escherichia coli.
 15. The method of claim 9 whereinthe at least two siderophore receptor polypeptides have molecularweights of between about 60 kDa and about 100 kDa as determined byseparation by sodium dodecyl-polyacrylamide gel electrophoresis.
 16. Themethod of claim 9 wherein the at least two porins have molecular weightsof between about 30 kDa and about 43 kDa as determined by separation bysodium dodecyl-polyacrylamide gel electrophoresis.
 17. The method ofclaim 9 wherein the animal is a bovine.